Lu Group Research 2014
241 | Yang Yu, Arnab Mukherjee, Mark J. Nilges, Parisa Hosseinzadeh, Kyle D. Miner, Yi Lu
"Direct EPR Observation of a Tyrosyl Radical in a Functional Oxidase Model in Myoglobin during both H2O2 and O2 Reactions"
J. Am. Chem. Soc. 136, (4) 1174-1177 (2014) (available online) [PDF] [doi]
240 | Hang Xing, Kevin Hwang, Ji Li, Seyed-Fakhreddin Torabi, Yi Lu
"DNA Aptamer Technology for Future Personalized Medicine"
Curr. Opin. Chem. Eng. 4, 79-87 (2014) [PDF] [doi]
239 | Hirotoshi Matsumura, Takahiro Hayashi, Saumen Chakraborty, Yi Lu, Pierre Moenne-Loccoz
"The production of nitrous oxide (N2O) by the heme/nonheme diiron center of engineered myoglobins (FeBMbs) proceeds through a trans iron-nitrosyl dimer"
J. Am. Chem. Soc. 136, (6) 2420-2431 (2014) (available online) [PDF] [doi]
238 | Igor D. Petrik, Jing Liu, Yi Lu
"Metalloenzyme Design and Engineering through Strategic Modifications of Native Protein Scaffolds"
Curr. Opin. Chem. Biol. 19, 67-75 (2014) [PDF] [doi]
237 | Ying-Wu Lin, Jiangyun Wang, Yi Lu
"Functional tuning and expanding of myoglobin by rational protein design"
Science China Chemistry 57, (3) 346-355 (2014) [PDF] [doi]
236 | Ilia Kaminker, Tiffany D Wilson, Masha G Savelieff; Yonatan Hovav, Herbert Zimmermann, Yi Lu, Daniella Goldfarb
"Correlating nuclear frequencies by two-dimensional ELDOR-detected NMR spectroscopy"
J. Mag. Reson. 240, 77-89 (2014) [PDF] [doi]
235 | Nathan A. Sieracki, Shiliang Tian, Ryan G. Hadt, Jun-Long Zhang, Julia S. Woertink, Mark J. Nilges, Furong Sun, Edward I. Solomon, Yi Lu
"Copper-sulfenate complex from oxidation of a cavity mutant of Pseudomonas aeruginosa azurin"
Proceedings of the National Academy of Science of the United States of America 111, (3) 924-929 (2014) [PDF] [doi]
234 | Jing Liu, Saumen Chakraborty, Parisa Hosseinzadeh, Yang Yu, Shiliang Tian, Igor Petrik, Ambika Bhagi, Yi Lu
"Metalloproteins Containing Cytochrome, Iron-sulfur or Copper Redox Centers"
Chem. Rev.(2014) (in press)
233 | Saumen Chakraborty, Julian Reed, Matthew Ross, Mark J. Nilges, Igor D. Petrik, Soumya Ghosh, Sharon Hammes-Schiffer, J. Timothy Sage, Yong Zhang, Charles E. Schulz, Yi Lu
"Spectroscopic and Computational Study of a Non-heme Iron-Nitrosyl Center in a Biosynthetic Model of Nitric Oxide Reductases in Myoglobin Containing Zn-protoporphyrin IX: Support for the Trans Mechanism of NO Reduction"
Angew. Chem., Int. Ed. 53, (9) 2417-2421 (2014) (available online) [PDF] [doi]
232 | Yu Xiang, Yi Lu
"DNA as Sensors and Imaging Agents for Metal Ions"
Inorganic Chemistry(2014) [PDF] [doi]
231 | Kevin M Clark, Yang Yu, Wilfred A van der Donk, Ninian Blackburn, Yi Lu
"Modulating the Copper-Sulfur Interaction in Type 1 Blue Copper Azurin by Replacing Cys112 with Nonproteinogenic Homocysteine"
Inorg. Chem. Frontiers 1, (2) 153-158 (2014) [PDF] [doi]
230 | Lauren A. Denofrio-Corrales, Antoinette Burton Yi Lu (Mary-Ann Winkelmes, Kyle Mays eds.)
"From Desk to Bench, Linking Students' Interests to Science Curricula"
Illinois Sampler: Teaching on the Prairie, 27-33 (2014) [archived PDF]
229 | Seyed-Fakhreddin Torabi, Yi Lu
"Functional DNA Nanomaterials for Sensing and Imaging in Living Cells"
Curr. Opin. Biotech. 28, 88-95 (2014) [PDF] [doi]
228 | Li Huey Tan, Hang Xing, Hongyu Chen, Yi Lu
"Facile and efficient preparation of anisotropic DNA-functionalized gold nanoparticles and their regioselective assembly"
Journal of the American Chemical Society(135) 17675-17678 (2013) [PDF] [doi]
227 | Longhua Tang, Ik Su Chun, Zidong Wang, Jinghong Li, Xiuling Li, Yi Lu
"DNA Detection using Plasmonic Enhanced Near-Infrared Photoluminescence of Gallium Arsenide"
Analytical Chemistry 85, (20) 9522-9527 (2013) [PDF] [doi]
226 | Yu Xiang, Peiwen Wu, Li Huey Tan, Yi Lu
"DNAzyme-Functionalized Gold Nanoparticles for Biosensing"
Advances in Biochemical Engineering/Biotechnology, Springer Berlin Heidelberg; pp. 1-28 (2013) [PDF] [doi]
225 | Ming-Li Tsai, Ryan G. Hadt, Nicholas M. Marshall, Tiffany D. Wilson, Yi Lu, Edward I. Solomon
"Axial interactions in the mixed-valent CuA active site and role of the axial methionine in electron transfer"
Proceedings of the National Academy of Sciences 110, 14658-14663 (2013) [PDF] [doi]

Within Cu-containing electron transfer active sites, the role of the axial ligand in type 1 sites is well defined, yet its role in the binuclear mixed-valent CuA sites is less clear. Recently, the mutation of the axial Met to Leu in a CuA site engineered into azurin (CuA Az) was found to have a limited effect on E0 relative to this mutation in blue copper (BC). Detailed low-temperature absorption and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance studies on CuA Az (WT) and its M123X (X = Q, L, H) axial ligand variants indicated stronger axial ligation in M123L/H. Spectroscopically validated density functional theory calculations show that the smaller ?E0 is attributed to H2O coordination to the Cu center in the M123L mutant in CuA but not in the equivalent BC variant. The comparable stabilization energy of the oxidized over the reduced state in CuA and BC (CuA   180 mV; BC   250 mV) indicates that the S(Met) influences E0 similarly in both. Electron delocalization over two Cu centers in CuA was found to minimize the JahnTeller distortion induced by the axial Met ligand and lower the inner-sphere reorganization energy. The CuS(Met) bond in oxidized CuA is weak (5.2 kcal/mol) but energetically similar to that of BC, which demonstrates that the protein matrix also serves an entatic role in keeping the Met bound to the active site to tune down E0 while maintaining a low reorganization energy required for rapid electron transfer under physiological conditions.
224 | Weichen Xu, Hang Xing, Yi Lu
"A smart T1-weighted MRI contrast agent for uranyl cations based on a DNAzyme-gadolinium conjugate"
Analyst 138, (21) 6266-6269 (2013) [archived PDF] [doi]

Rational design of smart MRI contrast agents with high specificity for metal ions remains a challenge. Here, we report a general strategy for the design of smart MRI contrast agents for detecting metal ions based on conjugation of a DNAzyme with a gadolinium complex. The 39E DNAzyme, which has high selectivity for UO22+, was conjugated to Gd(iii)-DOTA and streptavidin. The binding of UO22+ to its 39E DNAzyme resulted in the dissociation of Gd(iii)-DOTA from the large streptavidin, leading to a decrease of the T1 correlation time, and a change in the MRI signal.
223 | Tingting Chen, Yihui Hu, Yao Cen, Xia Chu, Yi Lu
"A Dual-Emission Fluorescent Nanocomplex of Gold Clusters Decorated Silica Particle for Live Cell Imaging of Highly Reactive Oxygen Species"
Journal of the American Chemical Society 135, 11595-11601 (2013) [PDF] [archived PDF] [doi]

A novel nanocomplex displaying single-excitation and dual-emission fluorescent properties has been developed through a crown-like assembly of dye-encapsulated silica particles decorated with satellite AuNCs for live cell imaging of highly reactive oxygen species (hROS), including OH, ClO- and ONOO-. The design of this nanocomplex is based on our new finding that the strong fluorescence of AuNCs can be sensitively and selectively quenched by these hROS. The nanocomplex is demonstrated to have excellent biocompatibility, high intracellular delivery efficiency, and stability for long-time observations. The results reveal that the nanocomplex provides a sensitive sensor for rapid imaging of hROS signaling with high selectivity and contrast.
222 | Marjorie Cepeda-Plaza, Eric L. Null, Yi Lu
"Metal Ion as Both a Cofactor and a Probe of Metal- binding Sites in a Uranyl-Specific DNAzyme: a Uranyl Photocleavage Study"
Nucleic Acids Res. 41, (20) 9361-9370 (2013) [PDF] [doi]
221 | Hang Xing, Li Tang, Xujuan Yang, Kevin Hwang, Wendan Wang, Qian Yin, Ngo Yin Wong, Lawrence W. Dobrucki, Norio Yasui, John Katzenellenbogen, William Helferich, Jianjun Cheng, Yi Lu
"Selective Delivery of an Anticancer Drug with Aptamer-Functionalized Liposomes to Breast Cancer Cells in Vitro and in Vivo"
J. Mater. Chem. B 1, (39) 5288-5297 (2013) [archived PDF] [doi]

Selective targeting of cancer cells is a critical step in cancer diagnosis and therapy. To address this need, DNA aptamers have attracted significant attention as possible targeting ligands. However, while their use in targeting cancer cells in vitro has been reported, their effectiveness has rarely been established in vivo. Here we report the development of a liposomal drug delivery system for targeted anticancer chemotherapy. Liposomes were prepared containing doxorubicin as a payload, and functionalized with AS1411, a DNA aptamer with strong binding affinity for nucleolin. AS1411 aptamer-functionalized liposomes increased cellular internalization and cytotoxicity to MCF-7 breast cancer cells as compared to non-targeting liposomes. Furthermore, targeted liposomal doxorubicin improved antitumor efficacy against xenograft MCF-7 breast tumors in athymic nude mice, attributable to their enhanced tumor tissue penetration. This study suggests that AS1411 aptamer-functionalized liposomes can recognize nucleolin overexpressed on MCF-7 cell surface, and therefore enable drug delivery with high specificity and selectivity.
220 | Hui Wei, Stephen House, Jiangjiexing Wu, Jiong Zhang, Zidong Wang, Ying He, Yi-Gui Gao, Howard Robinson, Wei Li, Jian-Min Zuo, Ian M. Robertson, Yi Lu
"Enhanced and Tunable Fluorescent Quantum Dots within a Single Crystal of Protein"
Nano Research 6, (9) 627-634 (2013) [PDF] [archived PDF] [doi]
219 | Ole Farver, Nicholas M. Marshall, Scot Wherland, Yi Lu, Israel Pecht
"Designed azurins show lower reorganization free energies for intraprotein electron transfer"
Proceedings of the National Academy of Sciences 110, 10536-10540 (2013) [PDF] [archived PDF] [doi]

Low reorganization free energies are necessary for fast electron transfer (ET) reactions. Hence, rational design of redox proteins with lower reorganization free energies has been a long-standing challenge, promising to yield a deeper understanding of the underlying principles of ET reactivity and to enable potential applications in different energy conversion systems. Herein we report studies of the intramolecular ET from pulse radiolytically produced disulfide radicals to Cu(II) in rationally designed azurin mutants. In these mutants, the copper coordination sphere has been fine-tuned to span a wide range of reduction potentials while leaving the metal binding site effectively undisrupted. We find that the reorganization free energies of ET within the mutants are indeed lower than that of WT azurin, increasing the intramolecular ET rate constants almost 10-fold: changes that are correlated with increased flexibility of their copper sites. Moreover, the lower reorganization free energy results in the ET rate constants reaching a maximum value at higher driving forces, as predicted by the Marcus theory.
218 | Le-Le Li, Mengying Xie, Jie Wang, Xinyang Li, Cheng Wang, Quan Yuan, Dai-Wen Pang, Yi Lu, Weihong Tan
"A vitamin-responsive mesoporous nanocarrier with DNA aptamer-mediated cell targeting"
Chem. Commun. 49, (52) 5823-5825 (2013) [archived PDF] [doi]

A smart drug delivery system with cancer cell targeting and bioresponsive controlled drug release has been constructed by taking advantage of a protein-capped mesoporous nanovalve and a DNA aptamer.
217 | Peiwen Wu, Kevin Hwang, Tian Lan, Yi Lu
"A DNAzyme-Gold Nanoparticle Probe for Uranyl Ion in Living Cells"
Journal of the American Chemical Society 135, 5254-5257 (2013) [PDF] [archived PDF] [doi]

DNAzymes have shown great promise as a general platform for detecting metal ions, as many metal-specific DNAzymes can be obtained using in vitro selection. While DNAzyme-based metal sensors have found many applications in the extracellular environment, no intracellular application of DNAzyme sensors has yet been reported. Here, we demonstrate a novel type of metal ion sensor for intracellular metal ion detection. The probe consists of a 13 nm gold nanoparticle (AuNP) core functionalized with a shell consisting of a uranyl-specific 39E DNAzyme whose enzyme strand contains a thiol at the 3' end for conjugation to the AuNP, and whose substrate strand is modified with a Cy3 fluorophore at the 5' end and a molecular quencher at the 3' end. In the absence of uranyl, the fluorescence of the Cy3 is quenched by both AuNP and the molecular quencher. In the presence of uranyl, the DNAzyme cleaves the fluorophore-labeled substrate strand, resulting in release of the shorter product strand containing the Cy3 and increased fluorescence. We demonstrate that this DNAzyme-AuNP probe can readily enter cells and can serve as a metal ion sensor within a cellular environment, making it the first demonstration of DNAzymes as intracellular metal ion sensors. Such a method can be generally applied to the detection of other metal ions using other DNAzymes selected through in vitro selection.
216 | Zidong Wang, Madurai Srinivasan Bharathi, Ramanarayan Hariharaputran, Hang Xing, Longhua Tang, Jinghong Li, Yong-Wei Zhang, Yi Lu
"pH-Dependent Evolution of Five-Star Gold Nanostructures: An Experimental and Computational Study"
ACS Nano 7, 2258-2265 (2013) [PDF] [archived PDF] [doi]

Dendritic structures, such as snowflakes, have been observed in nature in far-from-equilibrium growth conditions. Mimicking these structures at the nanometer scale can result in nanomaterials with interesting properties for applications, such as plasmonics and biosensors. However, reliable production and systematic fine-tuning morphologies of these nanostructures, with novel hierarchical or complex structures, along with theoretical understanding of these processes, are still major challenges in the field. Here, we report a new method of using pH to control HAuCl4 reduction by hydroxylamine for facile production of gold nanostructures with morphologies in various symmetries and hierarchies, both in solution and on solid surface. Of particular interest is the observation of five-star-like dendritic and hierarchical gold nanostructures under certain reaction conditions. Phase-field modeling was used to understand the growth and formation dynamics of the five-star and other gold complex nanostructures, and the results not only explained the experimental observations, but also predicted control of the nanostructural morphologies using both pH and hydroxylamine concentrations. In addition to revealing interesting growth dynamics in forming fascinating complex gold nanostructures, the present work provides a pH-directed morphology control method as a facile way to synthesize and fine-tune the morphology of hierarchical gold nanostructures.
215 | Ngo Yin Wong, Hang Xing, Li Huey Tan, Yi Lu
"Nano-Encrypted Morse Code: A Versatile Approach to Programmable and Reversible Nanoscale Assembly and Disassembly"
Journal of the American Chemical Society 135, 2931-2934 (2013) [PDF] [archived PDF] [doi] [news]

While much work has been devoted to nanoscale assembly of functional materials, selective reversible assembly of components in the nanoscale pattern at selective sites has received much less attention. Exerting such a reversible control of the assembly process will make it possible to fine-tune the functional properties of the assembly and to realize more complex designs. Herein, by taking advantage of different binding affinities of biotin and desthiobiotin toward streptavidin, we demonstrate selective and reversible decoration of DNA origami tiles with streptavidin, including revealing an encrypted Morse code NANO and reversible exchange of uppercase letter I with lowercase i. The yields of the conjugations are high (>90%), and the process is reversible. We expect this versatile conjugation technique to be widely applicable with different nanomaterials and templates.
214 | Le-Le Li, Peiwen Wu, Kevin Hwang, Yi Lu
"An Exceptionally Simple Strategy for DNA-Functionalized Up-Conversion Nanoparticles as Biocompatible Agents for Nanoassembly, DNA Delivery, and Imaging"
Journal of the American Chemical Society 135, 2411-2414 (2013) [PDF] [archived PDF] [doi]

Lanthanide-doped up-conversion nanoparticles (UCNPs) have shown promise in biomedical applications. However, as the UCNPs are normally capped with hydrophobic ligands, it remains challenging to prepare biocompatible UCNPs with specific molecular recognition capabilities. We herein report an exceptionally simple strategy to prepare uniform DNA-modified UCNPs as versatile bioprobes. The approach can directly convert as-prepared hydrophobic UCNPs into water-soluble DNA-UCNPs without any chemical modification of UCNPs or oligonucleotides. Furthermore, DNA molecules on the DNA-UCNPs retain their biorecognition ability, allowing programmable assembly of hybrid nanostructures. More importantly, we show that these DNA-UCNPs are capable of crossing cell membranes without the need of transfection agents, and their use as agents for bioimaging and DNA delivery are also demonstrated. Finally, DNA aptamer-conjugated UCNPs can be readily used for targeted imaging of cancer cells.
213 | Nicholas M. Marshall, Kyle D. Miner, Tiffany D. Wilson, Yi Lu
"Rational Design of Protein Cages for Alternative Enzymatic Functions"
in "Coordination Chemistry in Protein Cages: Principles, Design, and Applications", Takafumi Ueno, Yoshihito Watanabe, eds.; John Wiley & Sons; pp. 111-150 (2013) [PDF] [doi]
212 | LeLe Li, Yi Lu
"Functional DNA-Integrated Nanomaterials for Biosensing"
in "DNA Nanotechnology: From Structure to Function", Chunhai Fan (ed.), Springer-Verlag Berlin Heidelberg; pp. 227-305 (2013) [PDF] [doi]
211 | Yi Lu, Saumen Chakraborty, Kyle D. Miner, Tiffany D. Wilson, Arnab Mukherjee, Yang Yu, Jing Liu, Nicholas M. Marshall
"Metalloprotein Design"
in "Comprehensive Inorganic Chemistry II", Elsevier Science; pp. 565-593 (2013) [doi]
210 | Yu Xiang, Yi Lu
"An Invasive DNA Approach toward a General method for Portable Quantification of Metal Ions using a Personal Glucose Meter"
Chem. Commun. 49, (6) (Nucleic Acids: new life, new materials - joint Chem. Comm., Org. Biomol. Chem., and RSC Adv. web themed issue) 585-587 (2013) [archived PDF] [doi]

We report herein a general methodology for metal ion detection using low-cost, simple, and widely accessible personal glucose meters through an invasive DNA approach.
209 | Tiffany D. Wilson, Yang Yu, Yi Lu
"Understanding copper-thiolate containing electron transfer centers by incorporation of unnatural amino acids and the CuA center into the type 1 copper protein azurin"
Coordination Chemistry Reviews 257, 260 - 276 (2013) [archived PDF] [doi]

Highly covalent copper-thiolate bonds are salient features of ubiquitous type 1 (T1) blue copper and purple CuA electron transfer (ET) centers in proteins. These centers are found in a wide variety of proteins, each having its own electron transfer partners, requiring the centers to possess a broad range of reduction potentials to match those of their redox partners and to perform ET functions under various driving forces, all while maintaining high ET efficiency. Unraveling the secrets of the success realized by these ET centers has relied upon the expertise of many scientific disciplines and sub-disciplines, including inorganic chemistry, microbiology, biochemistry, and biophysical chemistry. Here, we review the contribution of protein engineering approachesnamely, the incorporation of unnatural amino acids and a biosynthetic CuA cofactor into the T1 copper protein azurinto advancing the current understanding of how the unique structures of T1 copper and CuA centers confer their proteins with efficient and tailored ET properties.
208 | Yu Xiang, Yi Lu
"Expanding Targets of DNAzyme-Based Sensors through Deactivation and Activation of DNAzymes by Single Uracil Removal: Sensitive Fluorescent Assay of Uracil-DNA Glycosylase"
Analytical Chemistry 84, 9981-9987 (2012) [PDF] [archived PDF] [doi]

Although deoxyribozymes (DNAzymes) have been widely used as biosensors for the detection of their cofactors and the targets of related aptazymes, it is desirable to expand their range of analytes to take advantage of the DNAzyme-based signal amplification for more sensitive detections. In this study, the activity of uracil-DNA glycosylase (UNG) was successfully detected and quantified by deoxyuridine-modified DNAzymes that underwent UNG-dependent deactivation or activation. In one design, the indispensable thymidine T2.1 in the 817 DNAzyme was replaced with a deoxyuridine, resulting in minimal change of the DNAzymes activity. Since UNG is capable of removing uracils from single- or double-stranded DNAs, the modified DNAzyme was deactivated when the uracil at the indispensable thymidine site was eliminated by UNG. In another design, introducing a deoxyuridine to the 3' position of the deoxycytidine C13 in the catalytic core of the same DNAzyme caused significant decrease of the activity. The removal of the interfering deoxyuridine by UNG, however, activated the DNAzyme. By monitoring the activity change of the DNAzymes through the fluorescence enhancement from the DNAzyme-catalyzed cleavage of DNA substrates labeled by a fluorophore and quencher pair, the UNG activity was measured based on UNG-dependent deactivation and activation of the DNAzymes. This method was found to be able to detect UNG activity as low as 0.0034 U/mL. Such a method can be applied to the detection of other nucleotide-modifying enzymes and expand the analyte range of DNAzyme-based biosensors.
207 | Yi Lu, Nicholas Marshall
"Redox Potential"
in "Encyclopedia of Biophysics", In Robers G. (Ed.), Vol. 4, Springer-Verlag Berlin Heidelberg; pp. 2207-2211 (2012)
206 | Yu Xiang, Zidong Wang, Hang Xing, Yi Lu
"Expanding DNAzyme functionality through enzyme cascades with applications in single nucleotide repair and tunable DNA-directed assembly of nanomaterials"
Chem. Sci. 4, (1) 398-404 (2012) [archived PDF] [doi]

Many biological functions require two or more enzymes working together in cascades. While many examples of protein and RNA enzyme cascades are known, few enzyme cascades containing solely DNAzymes have been reported. Herein we demonstrate the combination of an 8-17 DNAzyme with RNA cleavage activity and an E47 DNAzyme with DNA ligation activity to achieve a new function of single ribonucleotide repair in DNA while maintaining the integrity of the original DNA sequence, which is difficult for a single DNAzyme to achieve. In addition, this method is applied to modify the sequences of DNA strands immobilized on the surface of nanoparticles to control the DNA-directed assembly selectively and sequentially. Such an approach can be applied to other DNAzymes with different activities to expand the functions of DNAzymes and the scope of their applications.
205 | Kazuyo Shigeta, Ying He, Erick Sutanto, Somi Kang, An-Phong Le, Ralph G. Nuzzo, Andrew G. Alleyne, Placid M. Ferreira, Yi Lu, John A. Rogers
"Functional Protein Microarrays by Electrohydrodynamic Jet Printing"
Analytical Chemistry 84, 10012-10018 (2012) [PDF] [archived PDF] [doi]

This paper reports the use of advanced forms of electrohydrodynamic jet (e-jet) printing for creating micro- and nanoscale patterns of proteins on various surfaces ranging from flat silica substrates to structured plasmonic crystals, suitable for micro/nanoarray analysis and other applications in both fluorescent and plasmonic detection modes. The approaches function well with diverse classes of proteins, including streptavidin, IgG, fibrinogen, and y-globulin. Detailed study reveals that the printing process does not adversely alter the protein structure or function, as demonstrated in the specific case of streptavidin through measurements of its binding specificity to biotin-modified DNA. Multinozzle printing systems enable several types of proteins (up to four currently) to be patterned on a single substrate, in rapid fashion and with excellent control over spatial dimensions and registration. High-speed, pulsed operational modes allow large-area printing, with narrow statistical distributions of drop size and spacing in patterns that include millions of droplets. The process is also compatible with the structured surfaces of plasmonic crystal substrates to enable detection without fluorescence. These collective characteristics suggest potential utility of e-jet techniques in wide-ranging areas of biotechnology, where its compatibility with various biomaterials and substrates with different topographies and surface chemistries, and ability to form deposits that range from thick films to submonolayer coatings, derive from the remote, noncontacting physical material transfer mode of operation.
204 | Ryan G. Hadt, Ning Sun, Nicholas M. Marshall, Keith O. Hodgson, Britt Hedman, Yi Lu, Edward I. Solomon
"Spectroscopic and DFT Studies of Second-Sphere Variants of the Type 1 Copper Site in Azurin: Covalent and Nonlocal Electrostatic Contributions to Reduction Potentials"
Journal of the American Chemical Society 134, 16701-16716 (2012) [PDF] [archived PDF] [doi]

The reduction potentials (E0) of type 1 (T1) or blue copper (BC) sites in proteins and enzymes with identical first coordination spheres around the redox active copper ion can vary by  400 mV. Here, we use a combination of low-temperature electronic absorption and magnetic circular dichroism, electron paramagnetic resonance, resonance Raman, and SK-edge X-ray absorption spectroscopies to investigate a series of second-sphere variantsF114P, N47S, and F114N in Pseudomonas aeruginosa azurinwhich modulate hydrogen bonding to and protein-derived dipoles nearby the CuS(Cys) bond. Density functional theory calculations correlated to the experimental data allow for the fractionation of the contributions to tuning E0 into covalent and nonlocal electrostatic components. These are found to be significant, comparable in magnitude, and additive for active H-bonds, while passive H-bonds are mostly nonlocal electrostatic in nature. For dipoles, these terms can be additive to or oppose one another. This study provides a methodology for uncoupling covalency from nonlocal electrostatics, which, when coupled to X-ray crystallographic data, distinguishes specific local interactions from more long-range protein/active interactions, while affording further insight into the second-sphere mechanisms available to the protein to tune the E0 of electron-transfer sites in biology.
203 | Le-Le Li, Pinghua Ge, Paul R. Selvin, Yi Lu
"Direct Detection of Adenosine in Undiluted Serum Using a Luminescent Aptamer Sensor Attached to a Terbium Complex"
Analytical Chemistry 84, 7852-7856 (2012) [PDF] [archived PDF] [doi] [news]

Aptamers, single-stranded nucleic acids that can selectively bind to various target molecules, have been widely used for constructing biosensors. A major challenge in this field, however, is direct sensing of analytes in complex biological media such as undiluted serum. While progress has been made in developing an inhomogeneous assay by using a preseparation step to wash away the interferences within serum, a facile strategy for direct detection of targets in homogeneous unprocessed serum is highly desired. We herein report a turn-on luminescent aptamer biosensor for the direct detection of adenosine in undiluted and unprocessed serum, by taking advantage of a terbium chelate complex with long luminescence lifetime to achieve time-resolved detection. The sensor exhibits a detection limit of 60 uM adenosine while marinating excellent selectivity that is comparable to those in buffer. The approach demonstrated here can be applied for direct detection and quantification of a broad range of analytes in biological media by using other aptamers.
202 | Zidong Wang, Longhua Tang, Li Huey Tan, Jinghong Li, Yi Lu
"Discovery of the DNA "Genetic Code" for Abiological Gold Nanoparticle Morphologies"
Angewandte Chemie International Edition 51, 9078-9082 (2012) [archived PDF] [doi] [news]
201 | Le-Le Li, Qian Yin, Jianjun Cheng, Yi Lu
"Polyvalent Mesoporous Silica Nanoparticle-Aptamer Bioconjugates Target Breast Cancer Cells"
Advanced Healthcare Materials 1, 567-572 (2012) [archived PDF] [doi]

Spatiotemporal control over the delivery of therapeutic agents is an outstanding challenge to cancer treatment. By taking advantage of recent advances in DNA aptamer biology and mesoporous silica nanotechnology, we report a general approach to design and fabricate controlled release drug delivery systems that are able to effectively target cancer cells. Specifically, polyvalent mesoporous silica nanocarriers-aptamer bioconjugates were constructed; the high-surface-area nanoporous core allowed high drug loading and the surface-conjugated aptamer facilitated the nanoparticle targeting of nucleolin overexpressed MCF-7 cells. The efficient cancer-cell-specific fluorescent imaging and drug delivery of the bioconjugates outline the great potential for therapeutic applications.
200 | Hong Liu, Yu Xiang, Yi Lu, Richard M. Crooks
"Aptamer-Based Origami Paper Analytical Device for Electrochemical Detection of Adenosine"
Angewandte Chemie International Edition 51, 6925-6928 (2012) [archived PDF] [doi]
199 | Kyle D. Miner, Arnab Mukherjee, Yi-Gui Gao, Eric L. Null, Igor D. Petrik, Xuan Zhao, Natasha Yeung, Howard Robinson, Yi Lu
"A Designed Functional Metalloenzyme that Reduces O2 to H2O with Over One Thousand Turnovers"
Angewandte Chemie International Edition 51, 5589-5592 (2012) [archived PDF] [doi] [news]
198 | Le-Le Li, Ruobing Zhang, Leilei Yin, Kezhi Zheng, Weiping Qin, Paul R. Selvin, Yi Lu
"Biomimetic Surface Engineering of Lanthanide-Doped Upconversion Nanoparticles as Versatile Bioprobes"
Angewandte Chemie International Edition 51, 6121-6125 (2012) [archived PDF] [doi]
197 | JungHeon Lee, Zidong Wang, Yi Lu
"DNAzyme-Based Sensing for Metal Ions in Ocean Platform"
in "Molecular Biological Technologies for Ocean Sensing", Springer Protocols Handbooks, Sonia M. Tiquia-Arashiro, ed.; Humana Press: New York, NY; pp. 103-116 (2012) [archived PDF] [doi]
196 | Hang Xing, Ngo Yin Wong, Yu Xiang, Yi Lu
"DNA aptamer functionalized nanomaterials for intracellular analysis, cancer cell imaging and drug delivery"
Current Opinion in Chemical Biology 16, 429-435 (2012) [doi]
195 | Xiaohong Liu, Yang Yu, Cheng Hu, Wei Zhang, Yi Lu, Jiangyun Wang
"Significant Increase of Oxidase Activity through the Genetic Incorporation of a Tyrosine-Histidine Cross-Link in a Myoglobin Model of Heme-Copper Oxidase"
Angewandte Chemie International Edition 51, 4312-4316 (2012) [doi]
194 | Yu Xiang, Yi Lu
"Portable and Quantitative Detection of Protein Biomarkers and Small Molecular Toxins Using Antibodies and Ubiquitous Personal Glucose Meters"
Analytical Chemistry 84, 4174-4178 (2012) [PDF] [archived PDF] [doi] [news]

Developing portable and low-cost methods for quantitative detection of large protein biomarkers and small molecular toxins can play a significant role in controlling and preventing diseases or toxins outbreaks. Despite years of research, most current methods still require laboratory-based or customized devices that are not widely available to the general public for quantitative analysis. We have previously demonstrated the use of personal glucose meters (PGMs) and functional DNAs for the detection of many nonglucose targets. However, the range of targets detectable by functional DNAs is limited at the current stage. To expand the range of targets that can be detected by PGMs, we report here the use of antibodies in combination with sandwich and competitive assays for quantitative detection of protein biomarkers (PSA, with a detection limit of 0.4 ng/mL) and small molecular toxins (Ochratoxin A, with a detection limit of 6.8 ng/mL), respectively. In both assay methods, with invertase conjugates as the link, quantitative detection is achieved via the dependence between the concentrations of the targets in the sample and the glucose measured by PGMs. Given the wide availability of antibodies for numerous targets, the methods demonstrated here can expand the range of target detection by PGMs significantly.
193 | Weiqiao Zeng, Alexander Barabanschikov, Ningyan Wang, Yi Lu, Jiyong Zhao, Wolfgang Sturhahn, E. Ercan Alp, J. Timothy Sage
"Vibrational dynamics of oxygenated heme proteins"
Chem. Commun. 48, (51) 6340-6342 (2012) [archived PDF] [doi]

Advanced spectroscopic techniques coupled with DFT calculations reveal the vibrational dynamics of the iron in stable dioxygen complexes with myoglobin and with a mutant engineered to model the catalytic site of heme-copper oxidases. The unprecedented level of detail will constrain computational modelling of reactions with oxygen.
192 | Siu Yee New, Nicholas M. Marshall, T. S. Andy Hor, Feng Xue, Yi Lu
"Redox tuning of two biological copper centers through non-covalent interactions: same trend but different magnitude"
Chem. Commun. 48, (35) 4217-4219 (2012) [archived PDF] [doi]

The same non-covalent interactions previously found to affect the redox potential (Em) of the mononuclear T1 Cu protein azurin (Az) are shown to also fine-tune the Em of the dinuclear CuA center in the same Az protein scaffold. The effects of these mutations are in the same direction but with smaller magnitude in the CuA site, due to dissipation of the effects by the dinuclear CuA center.
191 | Panshu Song, Yu Xiang, Hang Xing, Zhaojuan Zhou, Aijun Tong, Yi Lu
"Label-Free Catalytic and Molecular Beacon Containing an Abasic Site for Sensitive Fluorescent Detection of Small Inorganic and Organic Molecules"
Analytical Chemistry 84, 2916-2922 (2012) [PDF] [archived PDF] [doi]

In this work, two methods with complementary features, catalytic and molecular beacon (CAMB) and label-free fluorescent sensors using an abasic site, have been combined into new label-free CAMB sensors that possess advantages of each method. The label-free method using a dSpacer-containing molecular beacon makes CAMB more cost-effective and less interfering with the catalytic activity, while CAMB allows the label-free method to use true catalytic turnovers for signal amplifications, resulting in a new label-free CAMB sensor for Pb2+ ion, with a detection limit of 3.8 nM while maintaining the same selectivity. Furthermore, by using CAMB to overcome the label-free methods limitation of requiring excess enzyme strands, a new label-free CAMB sensor using aptazyme is also designed to detect adenosine down to 1.4 M, with excellent selectivity over other nucleosides.
190 | Hannah E. Ihms, Yi Lu
"In Vitro Selection of Metal Ion-Selective DNAzymes"
in "Ribozymes", Methods in Molecular Biology, Vol. 848, Jorg S. Hartig, ed.; Humana Press: Hatfield, UK; pp. 297-316 (2012) [archived PDF] [doi]
189 | Yu Xiang, Yi Lu
"Using Commercially Available Personal Glucose Meters for Portable Quantification of DNA"
Analytical Chemistry 84, 1975-1980 (2012) [PDF] [archived PDF] [doi] [news]
188 | Hui Wei, Yi Lu
"Catalysis of Gold Nanoparticles within Lysozyme Single Crystals"
Chemistry - An Asian Journal 7, 680-683 (2012) [archived PDF] [doi]
187 | Kevin E. Nelson, Hannah E. Ihms, Debapriya Mazumdar, Peter J. Bruesehoff, Yi Lu
"The Importance of Peripheral Sequences in Determining the Metal Selectivity of an in Vitro-Selected Co2+-Dependent DNAzyme"
ChemBioChem 13, 381-391 (2012) [archived PDF] [doi]

DNAzymes are catalytically active DNA molecules that use metal cofactors for their enzymatic functions. While a growing number of DNAzymes with diverse functions and metal selectivities have been reported, the relationships between metal ion selectivity, conserved sequences and structures responsible for selectivity remain to be elucidated. To address this issue, we report biochemical assays of a family of previously reported in vitro selected DNAzymes. This family includes the clone 11 DNAzyme, which was isolated by positive and negative selection, and the clone 18 DNAzyme, which was isolated by positive selection alone. The clone 11 DNAzyme has a higher selectivity for Co2+ over Pb2+ compared with clone 18. The reasons for this difference are explored here through phylogenetic comparison, mutational analysis and stepwise truncation. A novel DNAzyme truncation method incorporated a nick in the middle of the DNAzyme to allow for truncation close to the nicked site while preserving peripheral sequences at both ends of the DNAzyme. The results demonstrate that peripheral sequences within the substrate binding arms, most notably the stem loop, loop II, are sufficient to restore its selectivity for Co2+ over Pb2+ to levels observed in clone 11. A comparison of these sequences' secondary structures and Co2+ selectivities suggested that metastable structures affect metal ion selectivity. The Co2+ selectivity of the clone 11 DNAzyme showed that the metal ion binding and selectivities of small, in vitro selected DNAzymes may be more complex than previously appreciated, and that clone 11 may be more similar to larger ribozymes than to other small DNAzymes in its structural complexity and behavior. These factors should be taken into account when metal-ion selectivity is required in rationally designed DNAzymes and DNAzyme-based biosensors.
186 | Hang Xing, Zidong Wang, Zhida Xu, Ngo Yin Wong, Yu Xiang, Gang Logan Liu, Yi Lu
"DNA-Directed Assembly of Asymmetric Nanoclusters Using Janus Nanoparticles"
ACS Nano 6, 802-809 (2012) [PDF] [archived PDF] [doi] [news]
185 | Tian Lan, Yi Lu
"Metal Ion-Dependent DNAzymes and Their Applications as Biosensors"
in "Interplay between Metal Ions and Nucleic Acids", Metal Ions in Life Sciences, Vol. 10, Astrid Sigel, Helmut Sigel, Roland K. O. Sigel, eds.; Springer Netherlands; pp. 217-248 (2012) [archived PDF] [doi]

Long considered to serve solely as the genetic information carrier, DNA has been shown in 1994 to be able to act as DNA catalysts capable of catalyzing a trans-esterification reaction similar to the action of ribozymes and protein enzymes. Although not yet found in nature, numerous DNAzymes have been isolated through in vitro selection for catalyzing many different types of reactions in the presence of different metal ions and thus become a new class of metalloenzymes. What remains unclear is how DNA can carry out catalysis with simpler building blocks and fewer functional groups than ribozymes and protein enzymes and how DNA can bind metal ions specifically to perform these functions. In the past two decades, many biochemical and biophysical studies have been carried out on DNAzymes, especially RNA-cleaving DNAzymes. Important insights have been gained regarding their metal-dependent activity, global folding, metal binding sites, and catalytic mechanisms for these DNAzymes. Because of their high metal ion selectivity, one of the most important practical applications for DNAzymes is metal ion detection, resulting in highly sensitive and selective fluorescent, colorimetric, and electrochemical sensors for a wide range of metal ions such as Pb 2+ , UO2 2 +, $$\rmPb^2+,\ UO^2+_2,$$ including paramagnetic metal ions such as Cu 2+ . This chapter summarizes recent progresses in in vitro selection of metal ion-selective DNAzymes, their biochemical and biophysical studies and sensing applications.
184 | Tiffany D. Wilson, Masha G. Savelieff, Mark J. Nilges, Nicholas M. Marshall, Yi Lu
"Kinetics of Copper Incorporation into a Biosynthetic Purple CuA Azurin: Characterization of Red, Blue, and a New Intermediate Species"
Journal of the American Chemical Society 133, 20778-20792 (2011) [PDF] [archived PDF] [doi]
183 | Dewain K. Garner, Lei Liang, David A. Barrios, Jun-Long Zhang, Yi Lu
"The Important Role of Covalent Anchor Positions in Tuning Catalytic Properties of a Rationally Designed MnSalen-Containing Metalloenzyme"
ACS Catalysis 1, 1083-1089 (2011) [PDF] [doi]
180 | Takahiro Hayashi, Kyle D. Miner, Natasha Yeung, Ying-Wu Lin, Yi Lu, Pierre Moënne-Loccoz
"Spectroscopic Characterization of Mononitrosyl Complexes in Heme–Nonheme Diiron Centers within the Myoglobin Scaffold (FeBMbs): Relevance to Denitrifying NO Reductase"
Biochemistry 50, 5939-5947 (2011) [PDF] [archived PDF] [doi]
179 | Ngo Yin Wong, Chuan Zhang, Li Huey Tan, Yi Lu
"Site-Specific Attachment of Proteins onto a 3D DNA Tetrahedron through Backbone-Modified Phosphorothioate DNA"
Small 7, 1427-1430 (2011) [archived PDF] [doi]
178 | Weichen Xu, Yi Lu
"A smart magnetic resonance imaging contrast agent responsive to adenosine based on a DNA aptamer-conjugated gadolinium complex"
Chem. Commun. 47, (17) 4998-5000 (2011) [archived PDF] [doi]

We report a general strategy for developing a smart MRI contrast agent for the sensing of small molecules such as adenosine based on a DNA aptamer that is conjugated to a Gd compound and a protein streptavidin. The binding of adenosine to its aptamer results in the dissociation of the Gd compound from the large protein, leading to decreases in the rotational correlation time and thus change of MRI contrast.
177 | Hui Wei, Zidong Wang, Jiong Zhang, Stephen House, Yi-Gui Gao, Limin Yang, Howard Robinson, Li Huey Tan, Hang Xing, Changjun Hou, Ian M. Robertson, Jian-Min Zuo, Yi Lu
"Time-dependent, protein-directed growth of gold nanoparticles within a single crystal of lysozyme"
Nature Nanotechnology 6, 93-97 (2011) [PDF] [archived PDF] [doi] [news]
176 | Nandini Nagraj, Yi Lu
"Chapter 5 Catalytic Nucleic Acid Biosensors for Environmental Monitoring"
in "Nucleic Acid Biosensors for Environmental Pollution Monitoring", The Royal Society of Chemistry; pp. 82-98 (2011) [archived PDF] [doi]

The discovery that nucleic acids can perform catalytic functions in addition to being genetic information carriers has opened doors to a new paradigm in chemistry and biology. It was thought previously that all biological enzymes were proteins, but discoveries made over the last 30 years have changed this perception. In this chapter the properties of RNAzymes, DNAzymes, and aptazymes are illustrated. The application of these novel catalytic molecules to environmental monitoring is discussed.
175 | Xiao-Bing Zhang, Rong-Mei Kong, Yi Lu
"Metal Ion Sensors Based on DNAzymes and Related DNA Molecules"
Annual Review of Analytical Chemistry 4, 105-128 (2011) [PDF] [archived PDF] [doi]
174 | Steven M Berry, Yi Lu
"Protein Structure Design and Engineering"
in "Encyclopedia of Life Sciences", John Wiley & Sons, Ltd: Chichester, UK; (2011) [archived PDF] [doi]

Protein structure design and engineering is a research endeavour in which proteins with predicted structure and function are constructed in the laboratory through rational design, combinatorial selection or combination of both approaches. It is built upon our knowledge about the structure and function of proteins and can be accomplished either from scratch (de novo design) or based on native scaffolds (redesign). The area of protein design is an exciting and rapidly growing field, advancing from the design of simple protein structures, to those that are more complicated and recently to the designs of functional proteins. Design of artificial proteins containing unnatural amino acids, backbone linkages or cofactors have also been reported, making it possible to prepare proteins with structural and functional properties beyond those of native proteins. These advances bring us closer to realising the dream of tailor-made artificial enzymes with high catalytic efficiency and selectivity for biotechnological and pharmaceutical applications.Key Concepts:Key Concepts: * Protein structure design and engineering is a research endeavour in which proteins with predicted structure and function are constructed in the laboratory. * The protein design field can be organised into two complimentary approaches: rational design and combinatorial selection of the desired protein. * Rational protein design strategies can involve designing a protein from scratch (de novo design) or redesigning a protein with native scaffolds to achieve new structures and functions. * Combinatorial approaches can involve sampling a large population of proteins to select the desired one or it can involve numerous rounds of randomised mutation followed by selection to fine-tune properties being selected for. * Techniques are available that allow the incorporation of unnatural moieties, such as unnatural amino acids, backbone linkages or cofactors, into proteins. * The field has made a big stride recently in designing functional proteins.
173 | Seyed-Fakhreddin Torabi, Yi Lu
"Small-molecule diagnostics based on functional DNA nanotechnology: a dipstick test for mercury"
Faraday Discuss. 149, (0) 125-135 (2011) [archived PDF] [doi]

Detecting small molecular targets such as metal ions is just as important as detecting large molecules such as DNA, RNA and proteins, but the field of metal ion sensors has not yet been well developed. A good example of a metal ion target is mercury, which is highly toxic, widely distributed in the environment and affects human health. To develop a diagnostic platform for metal ions, we demonstrate that functional DNA-linked gold nanoparticles (AuNPs) can quickly and simply detect and quantify Hg2+ ions in aqueous solution, with high sensitivity and selectivity over competing metal ions. A linker DNA molecule containing thymine residues and sequences complementary to the DNA on the AuNPs was designed to aggregate DNA-functionalized AuNPs. When Hg2+ ions were introduced into this system, they induced the linker DNA to fold by forming thymine-Hg2+-thymine bonds. The linker DNA's folding caused the AuNPs to rapidly disassemble, which caused a discernable color change in the solution from purple to red. The limit of detection for Hg2+ in the present method is 5.4 nM, which is below the 10 nM maximum contaminant level defined by the US Environmental Protection Agency (EPA) for drinking water. Our results show that this Hg2+ detection method has excellent selectivity over other divalent metal ions (e.g. Pb2+, Cu2+, Mn2+, Co2+, Zn2+, Cd2+, Mg2+, Ca2+, and Ba2+). This system has been converted into a dipstick test using lateral-flow devices, making it even more practical for point-of-care diagnostics.
172 | Kevin M. Clark, Yang Yu, Nicholas M. Marshall, Nathan A. Sieracki, Mark J. Nilges, Ninian J. Blackburn, Wilfred A. van der Donk, Yi Lu
"Transforming a Blue Copper into a Red Copper Protein: Engineering Cysteine and Homocysteine into the Axial Position of Azurin Using Site-Directed Mutagenesis and Expressed Protein Ligation"
Journal of the American Chemical Society 132, 10093-10101 (2010) [PDF] [archived PDF] [doi]

Interactions of the axial ligand with its blue copper center are known to be important in tuning spectroscopic and redox properties of cupredoxins. While conversion of the blue copper center with a weak axial ligand to a green copper center containing a medium strength axial ligand has been demonstrated in cupredoxins, converting the blue copper center to a red copper center with a strong axial ligand has not been reported. Here we show that replacing Met121 in azurin from Pseudomonas aeruginosa with Cys caused an increased ratio (RL) of absorption at 447 nm over that at 621 nm. Whereas no axial Cu-S(Cys121) interaction in Met121Cys was detectable by extended X-ray absorption fine structure (EXAFS) spectroscopy at pH 5, similar to what was observed in native azurin with Met121 as the axial ligand, the Cu-S(Cys121) interaction at 2.74 is clearly visible at higher pH. Despite the higher RL and stronger axial Cys121 interaction with Cu(II) ion, the Met121Cys variant remains largely a type 1 copper protein at low pH (with hyperfine coupling constant A? = 54 10-4 cm-1 at pH 4 and 5), or distorted type 1 or green copper protein at high pH (A? = 87 10-4 cm-1 at pH 8 and 9), attributable to the relatively long distance between the axial ligand and copper and the constraint placed by the protein scaffold. To shorten the distance between axial ligand and copper, we replaced Met121 with a nonproteinogenic amino acid homocysteine that contains an extra methylene group, resulting in a variant whose spectra (RL= 1.5, and A? = 180 10-4 cm-1) and Cu-S(Cys) distance (2.22 ) are very similar to those of the red copper protein nitrosocyanin. Replacing Met121 with Cys or homocysteine resulted in lowering of the reduction potential from 222 mV in the native azurin to 95 3 mV for Met121Cys azurin and 113 6 mV for Met121Hcy azurin at pH 7. The results strongly support the coupled distortion model that helps explain axial ligand tuning of spectroscopic properties in cupredoxins, and demonstrate the power of using unnatural amino acids to address critical chemical biological questions.
171 | Ying-Wu Lin, Natasha Yeung, Yi-Gui Gao, Kyle D. Miner, Lanyu Lei, Howard Robinson, Yi Lu
"Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity"
Journal of the American Chemical Society 132, 9970-9972 (2010) [PDF] [archived PDF] [doi]

A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called FeBMb(-His)). A high resolution (1.65 ) crystal structure of Cu(II)-CN--FeBMb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The FeBMb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-FeBMb(-His) and Fe(II)-FeBMb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N2O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-FeBMb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-FeBMb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.
170 | Jung Heon Lee, Ngo Yin Wong, Li Huey Tan, Zidong Wang, Yi Lu
"Controlled Alignment of Multiple Proteins and Nanoparticles with Nanometer Resolution via Backbone-Modified Phosphorothioate DNA and Bifunctional Linkers"
Journal of the American Chemical Society 132, 8906-8908 (2010) [PDF] [archived PDF] [doi]

Controlled alignment of streptavidin (STV), myoglobin, and nanoparticles with nanometer resolution has been achieved via backbone-modified phosphorothioate DNA and biotin- and maleimide-containing bifunctional linkers. Introducing triplet biotin modifications in three adjacent PSs significantly increased the STV conjugation yield. By placing phosphorothioate modifications at multiple positions of a double stranded DNA template, monomer, dimer, and trimer STV−DNA assemblies were formed with the STVs placed at controlled positions. The activity of the conjugated protein has been demonstrated by binding biotinylated AuNPs onto STV−DNA complexes, indicating the use of the system as a template for the formation of AuNP dimers and trimers with STVs separated by distances of 10−30 nm. Furthermore, a melting temperature experiment carried out with an STV−dsDNA assembly showed that the bifunctional-linker-modified PS−DNA system is much more stable than base-modified conjugation systems. This method allows for high yield, nanoscale-precision conjugation of multiple proteins to DNA. The linker can be designed to conjugate any proteins and nanomaterials specifically for a wide range of applications.
169 | Matthew S. Thorum, Cyrus A. Anderson, Jeremy J. Hatch, Andrew S. Campbell, Nicholas M. Marshall, Steven C. Zimmerman, Yi Lu, Andrew A. Gewirth
"Direct, Electrocatalytic Oxygen Reduction by Laccase on Anthracene-2-methanethiol-Modified Gold"
The Journal of Physical Chemistry Letters 1, 2251-2254 (2010) [PDF] [archived PDF] [doi]

Laccase, a multicopper oxidase, catalyzes the four-electron reduction of oxygen to water. Upon adsorption to an electrode surface, laccase is known to reduce oxygen at overpotentials lower than the best noble metal electrocatalysts usually employed. Whereas the electrocatalytic activity of laccase is well established on carbon electrodes, laccase does not typically adsorb to better defined noble metal surfaces in an orientation that allows for efficient electrocatalysis. In this work, we utilized anthracene-2-methanethiol (AMT) to modify the surface of Au electrodes and examined the electrocatalytic activity of adsorbed laccase. AMT facilitated the adsorption of laccase, and the onset of electrocatalytic oxygen reduction was observed as high as 1.13VRHE. We observed linear Tafel behavior with a 144 mV/dec slope, consistent with an outer sphere single electron transfer from the electrode to a Cu site in the enzyme as the rate-determining step of the oxygen reduction mechanism.
168 | Jung Heon Lee, Mehmet V. Yigit, Debapriya Mazumdar, Yi Lu
"Molecular diagnostic and drug delivery agents based on aptamer-nanomaterial conjugates"
Advanced Drug Delivery Reviews 62, 592-605 (2010) [archived PDF] [doi]

Recent progress in an emerging area of designing aptamer and nanomaterial conjugates as molecular diagnostic and drug delivery agents in biomedical applications is summarized. Aptamers specific for a wide range of targets are first introduced and compared to antibodies. Methods of integrating these aptamers with a variety of nanomaterials, such as gold nanoparticles, quantum dots, carbon nanotubes, and superparamagnetic iron oxide nanoparticles, each with unique optical, magnetic, and electrochemical properties, are reviewed. Applications of these systems as fluorescent, colorimetric, magnetic resonance imaging, and electrochemical sensors in medical diagnostics are given, along with new applications as smart drug delivery agents.
167 | Jinli Zhang, Lin Zheng, Xian Wang, Ying Xiao, Yi Lu, Wei Li
"Branched silica nanostructures oriented by dynamic G-quadruplex transformation"
Materials Research Bulletin 45, 1954-1959 (2010) [archived PDF] [doi]

Polymorphic G-quadruplexes including stacked G-quartet planes are potentially promising DNA templates beyond Watson-Crick duplexes to construct inorganic nanostructures. In this work, a novel G-rich oligonucleotide that can be modulated from the antiparallel G-quadruplex to multi-stranded supermolecular assemblies was designed. Based on this dynamic G-quadruplex transformation, an interesting leaf-vein silica nanostructure with regular branched intervals was constructed. The G-quadruplex assembly structure was found to play a significant role in the organization of the silica structures.
166 | Xiao-Bing Zhang, Zidong Wang, Hang Xing, Yu Xiang, Yi Lu
"Catalytic and Molecular Beacons for Amplified Detection of Metal Ions and Organic Molecules with High Sensitivity"
Analytical Chemistry 82, 5005-5011 (2010) [PDF] [archived PDF] [doi]

The catalytic beacon has emerged as a general platform for sensing metal ions and organic molecules. However, few reports have taken advantage of the true potential of catalytic beacons in signal amplification through multiple enzymatic turnovers, as existing designs require either equal concentrations of substrate and DNAzyme or an excess of DNAzyme in order to maintain efficient quenching, eliminating the excess of substrate necessary for multiple turnovers. On the basis of the large difference in the melting temperatures between the intramolecular molecular beacon stem and intermolecular products of identical sequences, we here report a general strategy of catalytic and molecular beacon (CAMB) that combines the advantages of the molecular beacon for highly efficient quenching with the catalytic beacon for amplified sensing through enzymatic turnovers. Such a CAMB design allows detection of metal ions such as Pb2+ with a high sensitivity (LOD = 600 pM). Furthermore, the aptamer sequence has been introduced into DNAzyme to use the modified CAMB for amplified sensing of adenosine with similar high sensitivity. These results together demonstrate that CAMB provides a general platform for amplified detection of a wide range of targets.
165 | Tian Lan, Kimberly Furuya, Yi Lu
"A highly selective lead sensor based on a classic lead DNAzyme"
Chemical Communications 46, 3896-3898 (2010) [archived PDF] [doi]

A catalytic beacon sensor for Pb2+ has been developed based on the first DNAzyme discovered in the field, and such a sensor has shown a much higher metal ion selectivity (40000 times) than the previously reported Pb2+ sensor based on 8-17 DNAzyme and thus is suitable for a wider range of practical applications.
164 | Zidong Wang, Jieqian Zhang, Jonathan M. Ekman, Paul J. A. Kenis, Yi Lu
"DNA-Mediated Control of Metal Nanoparticle Shape: One-Pot Synthesis and Cellular Uptake of Highly Stable and Functional Gold Nanoflowers"
Nano Letters 10, 1886-1891 (2010) [PDF] [archived PDF] [doi]

The effects of different DNA molecules of the same length on the morphology of gold nanoparticles during synthesis are investigated. While spherical nanoparticles (AuNS) are observed in the presence of 30-mer poly T, like that in the absence of DNA, 30-mer poly A or poly C induces formation of the flower-shaped gold nanoparticle (AuNF). Detailed mechanistic studies indicate that the difference in DNA affinity to the AuNP plays a major role in the different morphology control processes. The DNA adsorbed on the AuNS surface could act as template to mediate the formation of flower-like gold nanoparticles. The formation of the AuNF can result from either selective deposition of the reduced gold metal on AuNS templated by surface bound DNA or uneven growth of the AuNS due to the binding of DNA to the surface. Furthermore, DNA functionalization with high stability was realized in situ during the one-step synthesis while retaining their biorecognition ability, allowing programmable assembly of new nanostructures. We have also shown that the DNA-functionalized nanoflowers can be readily uptaken by cells and visualized under dark-field microscopy.
163 | Hui Wei, Zidong Wang, Limin Yang, Shiliang Tian, Changjun Hou, Yi Lu
"Lysozyme-stabilized gold fluorescent cluster: Synthesis and application as Hg2+ sensor"
The Analyst 135, 1406-1410 (2010) [archived PDF] [doi]

Highly fluorescent gold clusters have been synthesized in basic aqueous solution by using lysozyme as reducing and stabilizing agents. The lysozyme-stabilized gold fluorescent clusters (LsGFC) have an average size of 1 nm and emission [similar] 657 nm. The fluorescence could be specifically quenched by Hg2+, so the LsGFC can be used as a sensor for sensitive and selective Hg2+ detection with a detection limit of 10 nM.
162 | Masha Savelieff, Yi Lu
"CuA centers and their biosynthetic models in azurin"
Journal of Biological Inorganic Chemistry 15, 461-483 (2010) [archived PDF] [doi]

Abstract  CuA is a binuclear copper center that functions as an electron transfer agent, cycling between a reduced Cu(I)Cu(I) state and an oxidized mixed-valence Cu(+1.5)\textperiodcentered\textperiodcentered\textperiodcenteredCu(+1.5) state. The copper ions are bridged by two cysteine thiolate ligands and form a copper--copper bond, the first reported of its kind in Nature. Such a ``diamond-core'' Cu2S(Cys)2 structure allows an unpaired electron to be completely delocalized over the two copper ions and contributes to its highly efficient electron transfer properties. This review provides accounts of how the CuA center was structurally characterized and highlights its salient spectroscopic properties. In the process, it introduces the CuA center in four different systems---native protein systems, soluble protein truncates of native proteins, synthetic models using organic molecules, and biosynthetic models using proteins as ligands---with a greater emphasis on biosynthetic models of CuA, especially on new, deeper insights gained from their studies.
161 | Ying-Wu Lin, Natasha Yeung, Yi-Gui Gao, Kyle D. Miner, Shiliang Tian, Howard Robinson, Yi Lu
"Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin"
Proceedings of the National Academy of Sciences 107, 8581-8586 (2010) [PDF] [archived PDF] [doi]

A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative FeB site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by ∼110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a ∼100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the FeB site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.
160 | Yu Xiang, Zidong Wang, Hang Xing, Ngo Yin Wong, Yi Lu
"Label-Free Fluorescent Functional DNA Sensors Using Unmodified DNA: A Vacant Site Approach"
Analytical Chemistry 82, 4122-4129 (2010) [PDF] [archived PDF] [doi]

A general methodology to design label-free fluorescent functional DNA sensors using unmodified DNA via a vacant site approach is described. By extending one end of DNA with a loop, a vacant site that binds an extrinsic fluorophore, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), could be created at a selected position in the DNA duplex region of DNAzymes or aptamers. When the vacant site binds ATMND, ATMND’s fluorescence is quenched. This fluorescence can be recovered when one strand of the duplex DNA is released through either metal ion-dependent cleavage by DNAzymes or analyte-dependent structural-switching by aptamers. Through this design, label-free fluorescent sensors for Pb2+, UO22+, Hg2+, and adenosine have been successfully developed. These sensors have high selectivity and sensitivity; detection limits as low as 3 nM, 8 nM, 30 nM, and 6 μM have been achieved for UO22+, Pb2+, Hg2+ and adenosine, respectively. Control experiments using vacant-site-free DNA duplexes and inactive variants of the functional DNAs indicate that the presence of the vacant site and the activity of the functional DNAs are essential for the performance of the proposed sensors. The vacant site approach demonstrated here can be used to design many other label-free fluorescent sensors to detect a wide range of analytes.
159 | Yi Lu
"Metal ions as matchmakers for proteins"
Proceedings of the National Academy of Sciences 107, 1811-1812 (2010) [PDF] [archived PDF] [doi]
158 | Weichen Xu, Yi Lu
"Label-Free Fluorescent Aptamer Sensor Based on Regulation of Malachite Green Fluorescence"
Analytical Chemistry 82, 574-578 (2010) [PDF] [archived PDF] [doi]

We report a label-free fluorescent aptamer sensor for adenosine based on the regulation of malachite green (MG) fluorescence, with comparable sensitivity and selectivity to other labeled adenosine aptamer-based sensors. The sensor consists of free MG, an aptamer strand containing an adenosine aptamer next to an MG aptamer, and a bridging strand that partially hybridizes to the aptamer strand. Such a hybridization prevents MG from binding to MG aptamer, resulting in low fluorescence of MG in the absence of adenosine. Addition of adenosine causes the adenosine aptamer to bind adenosine, weakening the hybridization of the aptamer strand with the bridging strand, making it possible for MG to bind to the aptamer strand and exhibit high fluorescence intensity. Since this design is based purely on nucleic acid hybridization, it can be generally applied to other aptamers for the label-free detection of a broad range of analytes.
157 | Changyuan Lu, Xuan Zhao, Yi Lu, Denis L. Rousseau, Syun-Ru Yeh
"Role of Copper Ion in Regulating Ligand Binding in a Myoglobin-Based Cytochrome c Oxidase Model"
Journal of the American Chemical Society 132, 1598-1605 (2010) [PDF] [archived PDF] [doi]

Cytochrome c oxidase (CcO), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the four-electron reduction of dioxygen to water in a binuclear center comprised of a high-spin heme (heme a3) and a copper atom (CuB) coordinated by three histidine residues. As a minimum model for CcO, a mutant of sperm whale myoglobin, named CuBMb, has been engineered, in which a copper atom is held in the distal heme pocket by the native E7 histidine and two nonnative histidine residues. In this work, the role of the copper in regulating ligand binding in CuBMb was investigated. Resonance Raman studies show that the presence of copper in CO-bound CuBMb leads to a CcO-like distal heme pocket. Stopped-flow data show that, upon the initiation of the CO binding reaction, the ligand first binds to the Cu+; it subsequently transfers from Cu+ to Fe2+ in an intramolecular process, similar to that reported for CcO. The high CO affinity toward Cu+ and the slow intramolecular CO transfer rate between Cu+ and Fe2+ in the CuBMb/Cu+ complex are analogous to those in Thermus thermophilus CcO (TtCcO) but distinct from those in bovine CcO (bCcO). Additional kinetic studies show that, upon photolysis of the NO-bound CuBMb/Cu+ complex, the photolyzed ligand transiently binds to Cu+ and subsequently rebinds to Fe2+, accounting for the 100% geminate recombination yield, similar to that found in TtCcO. The data demonstrate that the CuBMb/Cu+ complex reproduces essential structural and kinetic features of CcO and that the complex is more akin to TtCcO than to bCcO.
156 | Debapriya Mazumdar, Juewen Liu, Geng Lu, Juanzuo Zhou, Yi Lu
"Easy-to-use dipstick tests for detection of lead in paints using non-cross-linked gold nanoparticle-DNAzyme conjugates"
Chemical Communications 46, 1416-1418 (2010) [archived PDF] [doi] [news]

Easy-to-use dipstick tests for lead have been developed by immobilizing nanoparticle-DNAzyme conjugates on lateral flow devices and their application for detecting lead in paints is demonstrated.
155 | Eric L. Null, Yi Lu
"Rapid determination of enantiomeric ratio using fluorescent DNA or RNA aptamers"
The Analyst 135, 419-422 (2010) [archived PDF] [doi]

The natural chirality of DNA and RNA aptamers has been used to develop fluorescent agents to determine the enantiomeric ratio of adenosine and arginine, respectively. The quantification is based on structure-switching DNA or RNA aptamers labeled with fluorophore and quencher, allowing chiral detection down to 0.1:99.9 (L:D) for arginine after calibration. Such a method provides a general platform for simple, low-cost and high throughput detection and quantification of chirality of a broad range of molecules.
154 | Yu Xiang, Aijun Tong, Yi Lu
"Abasic Site-Containing DNAzyme and Aptamer for Label-Free Fluorescent Detection of Pb2+ and Adenosine with High Sensitivity, Selectivity, and Tunable Dynamic Range"
Journal of the American Chemical Society 131, 15352-15357 (2009) [PDF] [doi] [news]

An abasic site called dSpacer has been introduced into duplex regions of the 8−17 DNAzyme and adenosine aptamer for label-free fluorescent detection of Pb2+ and adenosine, respectively. The dSpacer can bind an extrinsic fluorescent compound, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), and quench its fluorescence. Addition of Pb2+ enables the DNAzyme to cleave its substrate and release ATMND from DNA duplex, recovering the fluorescence of ATMND. Similarly, the presence of adenosine induces structural switching of the aptamer, resulting in the release of ATMND from the DNA duplex and a subsequent fluorescence enhancement. Under optimized conditions, this label-free method exhibits detection limits of 4 nM for Pb2+ and 3.4 μM for adenosine, which are even lower than those of the corresponding labeled-DNAzyme and aptamer sensors. These low detection limits have been obtained without compromising any of the selectivity of the sensors. Finally, the dynamic range of the adenosine sensor has been tuned by varying the number of hybridized base-pairs in the aptamer duplex. The method demonstrated here can be applied for label-free detection and quantification of a broad range of analytes using other DNAzymes and aptamers.
153 | Natasha Yeung, Ying-Wu Lin, Yi-Gui Gao, Xuan Zhao, Brandy S. Russell, Lanyu Lei, Kyle D. Miner, Howard Robinson, Yi Lu
"Rational design of a structural and functional nitric oxide reductase"
Nature 462, 1079-1082 (2009) [doi] [news]
152 | Nicholas M. Marshall, Dewain K. Garner, Tiffany D. Wilson, Yi-Gui Gao, Howard Robinson, Mark J. Nilges, Yi Lu
"Rationally tuning the reduction potential of a single cupredoxin beyond the natural range"
Nature 462, 113-116 (2009) [doi] [news]
151 | Mehmet Veysel Yigit, Abhijit Mishra, Rong Tong, Jianjun Cheng, Gerard C.L. Wong, Yi Lu
"Inorganic Mercury Detection and Controlled Release of Chelating Agents from Ion-Responsive Liposomes"
Chemistry & Biology 16, 937-942 (2009) [doi]

release profile is particularly useful insituations in which the local levels of Hg contamination vary, or if such contamination is time dependent.
150 | Yi Lu, Natasha Yeung, Nathan Sieracki, Nicholas M. Marshall
"Design of functional metalloproteins"
Nature 460, 855-862 (2009) [doi]
149 | Zhengyi Wu, Qin Liu, Xiao Liang, Xiaoliang Yang, Ningyan Wang, Xinghao Wang, Hongzhe Sun, Yi Lu, Zijian Guo
"Reactivity of platinum-based antitumor drugs towards a Met- and His-rich 20mer peptide corresponding to the N-terminal domain of human copper transporter 1"
Journal of Biological Inorganic Chemistry 14, 1313-1323 (2009) [doi]

Abstract  Cellular uptake of platinum-based antitumor drugs is a critical step in the mechanism of the drug action and associated resistance, and deeper understanding of this step may inspire development of novel methods for new drugs with reduced resistance. Human copper transporter 1 (hCtr1), a copper influx protein, was recently found to facilitate the cellular entry of several platinum drugs. In the work reported here, we constructed a Met- and His-rich 20mer peptide (hCtr1-N20) corresponding to the N-terminal domain of hCtr1, which is the essential domain of hCtr1 for transporting platinum drugs. The interactions of the peptide with cisplatin and its analogues, including transplatin, carboplatin, oxaliplatin, and [Pt(l-Met)Cl2], were explored at the molecular level. Electrospray ionization (ESI) mass spectrometry (MS) data revealed that all of the platinum(II) complexes used in present study can bind to hCtr1-N20 in 1:1 and 2:1 stoichiometry. Four Met residues should be involved in binding to cis-platinum complexes on the basis of the tandem MS spectrometry and previously reported data. Time-dependent 2D [1H,15N] heteronuclear single quantum coherence NMR spectra indicate the reaction of cisplatin with hCtr1-N20 is a stepwise process. The intermediate, however, is transient, which is consistent with the ESI-MS results. Time-dependent ESI-MS data revealed that the geometry and the properties of both the leaving and the nonleaving groups of platinum(II) complexes play essential roles in controlling the reactivity and formation of the final products with hCtr1-N20.
148 | Zehui Cao, Rong Tong, Abhijit Mishra, Weichen Xu, Gerard C L Wong, Jianjun Cheng, Yi Lu
"Reversible Cell-Specific Drug Delivery with Aptamer-Functionalized Liposomes"
Angewandte Chemie International Edition 48, 6494-6498 (2009) [doi] [news]

Aptamer advantages: Cell-specific delivery of the anticancer drug cisplatin through a nucleolin-aptamer-conjugated, cisplatin-encapsulating liposome delivery system is described. Calcein was incorporated into the target MCF-7 cells (see top image) but not into LNCaP cells (see bottom image). More importantly, the extent of delivery can be controlled by using a complementary DNA of the aptamer as an antidote.
147 | Nandini Nagraj, Juewen Liu, Stephanie Sterling, Jenny Wu, Yi Lu
"DNAzyme catalytic beacon sensors that resist temperature-dependent variations"
Chemical Communications 2009, 4103-4105 (2009) [doi]

The temperature-dependent variability of a Pb2+-specific 8-17E DNAzyme catalytic beacon sensor has been addressed through the introduction of mismatches in the DNAzyme, and the resulting sensors resist temperature-dependent variations from 4 to 30 [degree]C.
146 | Juewen Liu, Zehui Cao, Yi Lu
"Functional Nucleic Acid Sensors"
Chemical Reviews 109, 1948-1998 (2009) [doi]

145 | Jianhui Zhu, Miaoxin Lin, Damin Fan, Ziyi Wu, Yuncong Chen, Junfeng Zhang, Yi Lu, Zijian Guo
"The role of bridging ligands in determining DNA-binding ability and cross-linking patterns of dinuclear platinum(ii) antitumour complexes"
Dalton Transactions 2009, 10889-10895 (2009) [archived PDF] [doi]

The DNA binding ability and binding mode of platinum complexes are crucial factors that govern their cytotoxic activity. In this work, circular dichroism spectroscopy, gel electrophoresis and MALDI-TOF MS spectrometry combined with enzymatic degradation have been used to elucidate the role of bridging ligands in DNA-binding ability and cross-linking patterns of two dinuclear antitumour active platinum(ii) complexes, [cis-Pt(NH3)2Cl]2L1(NO3)2 (1, L1= 4,4[prime or minute]-methylenedianiline) and [cis-Pt(NH3)2Cl]2L2(NO3)2 (2, L2 = [small alpha],[small alpha][prime or minute]-diamino-p-xylene). Although both complexes have two cis-diammine-Pt(ii) moieties (1,1/c,c), complex 1 exhibits much higher DNA-binding ability than complex 2. The former readily forms both 1,3- and 1,4-intrastrand cross-links with DNA oligonucleotides, while the latter preferentially forms 1,4- rather than 1,3-intrastrand cross-links. Cytotoxicity studies against a human non-small-cell lung cancer cell line (A549) demonstrate that complex 1 has higher activity than 2. These results show that the linker properties play a critical role in controlling the DNA-binding and cross-linking abilities and in modulating the cytotoxicity of dinuclear platinum complexes.
144 | Debapriya Mazumdar, Nandini Nagraj, Hee-Kyung Kim, Xiangli Meng, Andrea K. Brown, Qian Sun, Wei Li, Yi Lu
"Activity, Folding and Z-DNA Formation of the 8-17 DNAzyme in the Presence of Monovalent Ions"
Journal of the American Chemical Society 131, 5506-5515 (2009) [PDF] [doi]

The effect of monovalent ions on both the reactivity and global folding of the 8-17 DNAzyme is investigated, and the results are compared with those of the hammerhead ribozyme, which has similar size and secondary structure. In contrast to the hammerhead ribozyme, the 8-17 DNAzyme activity is not detectable in the presence of 4 M K+, Rb+, or Cs+ or in the presence of 80 mM, [Co(NH3)6]3+. Only 4 M Li+, NH4+ and, to a lesser extent, Na+ conferred detectable activity. The observed rate constants (kobs ≈ 10−3 min−1 for Li+ and NH4+) are ∼1000-fold lower than that in the presence of 10 mM Mg2+, and ∼200,000-fold slower than that in the presence of 100 μM Pb2+. Since the hammerhead ribozyme displays monovalent ion-dependent activity that is often within ∼10-fold of divalent metal ion-dependent activity, these results suggest that the 8-17 DNAzyme, obtained by in vitro selections, has evolved to have a more stringent divalent metal ion requirement for high activity as compared to the naturally occurring ribozymes, making the 8-17 DNAzyme an excellent choice as a Pb2+ sensor with high selectivity. In contrast to the activity data, folding was observed in the presence of all the monovalent ions investigated, although those monovalent ions that do not support DNAzyme activity have weaker binding affinity (Kd ∼ 0.35 M for Rb+ and Cs+), while those that confer DNAzyme activity possess stronger affinity (Kd ∼ 0.22 M for Li+, Na+ and NH4+). In addition, a correlation between metal ion charge density, binding affinity and enzyme activity was found among mono- and divalent metal ions except Pb2+; higher charge density resulted in stronger affinity and higher activity, suggesting that the observed folding and activity is at least partially due to electrostatic interactions between ions and the DNAzyme. Finally, circular dichroism (CD) study has revealed Z-DNA formation with the monovalent metal ions, Zn2+ and Mg2+; the Kd values obtained using CD were in the same range as those obtained from folding studies using FRET. However, Z-DNA formation was not observed with Pb2+. These results indicate that Pb2+-dependent function follows a different mechanism from the monovalent metal ions and other divalent metal ions; in the presence of latter metal ions, metal-ion dependent folding and structural changes, including formation of Z-DNA, play an important role in the catalytic function of the 8-17 DNAzyme.
143 | Jun-Long Zhang, Dewain K Garner, Lei Liang, David A Barrios, Yi Lu
"Noncovalent Modulation of pH-Dependent Reactivity of a Mn-Salen Cofactor in Myoglobin with Hydrogen Peroxide"
Chemistry - A European Journal 15, 7481-7489 (2009) [doi]

To demonstrate protein modulation of metal-cofactor reactivity through noncovalent interactions, pH-dependent sulfoxidation and 2,2prime-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) oxidation reactivity of a designed myoglobin (Mb) containing non-native Mn-salen complex (1) was investigated using H2O2 as the oxidant. Incorporation of 1 inside the Mb resulted in an increase in the turnover numbers through exclusion of water from the metal complex and prevention of Mn-salen dimer formation. Interestingly, the presence of protein in itself is not enough to confer the increase activity as mutation of the distal His64 in Mb to Phe to remove hydrogen-bonding interactions resulted in no increase in the turnover numbers, while mutation His64 to Arg, another residue with ability to hydrogen-bond interactions, resulted in an increase in reactivity. These results strongly suggest that the distal ligand His64, through its hydrogen-bonding interaction, plays important roles in enhancing and fine-tuning reactivity of the Mn-salen complex. Nonlinear least-squares fitting of rate versus pH plots demonstrates that 1sdotMb(H64X) (X=H, R and F) and the control Mn-salen 1 exhibit pKa values varying from pH?6.4 to 8.3, and that the lower pKa of the distal ligand in 1sdotMb(H64X), the higher the reactivity it achieves. Moreover, in addition to the pKa at high pH, 1sdotMb displays another pKa at low pH, with pKa of 5.0\textpm0.08. A comparison of the effect of different pH on sulfoxidation and ABTS oxidation indicates that, while the intermediate produced at low pH conditions could only perform sulfoxidation, the intermediate at high pH could oxidize both sulfoxides and ABTS. Such a fine-control of reactivity through hydrogen-bonding interactions by the distal ligand to bind, orient and activate H2O2 is very important for designing artificial enzymes with dramatic different and tunable reactivity from catalysts without protein scaffolds.
142 | Danqun Huo, Limin Yang, Changjun Hou, Huanbao Fa, Xiaogang Luo, Yi Lu, Xiaolin Zheng, Jun Yang, Li Yang
"Molecular interactions of monosulfonate tetraphenylporphyrin (TPPS1) and meso-tetra(4-sulfonatophenyl)porphyrin (TPPS) with dimethyl methylphosphonate (DMMP)"
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 74, 336-343 (2009) [archived PDF] [doi]

The molecular interactions of monosulfonate tetraphenylporphyrin (TPPS1) and meso-tetra(4-sulfonatophenyl)porphyrin (TPPS) with dimethyl methylphosphonate (DMMP) have been investigated by UV-vis and fluorescence spectroscopies. The association constants and interaction stoichiometries of the bindings were obtained through Benesi-Hildebrand (B-H) method. Particularly, both linear and nonlinear fitting procedures were performed to evaluate the possible 1:2 interactions. Experimental results showed that hydrogen-bonding interactions existed in both of the two systems, resulting in regular changes in the absorbance and fluorescence characteristics of the porphyrins. The association constants and stoichiometries determined from absorbance and fluorescence studies were in excellent agreement. Using a nonlinear fitting method, we demonstrated that the one-step 1:2 interaction took place in the TPPS1-DMMP system, and the association constants were determined to be 71.4M-1 by absorbance measurements and 70.92M-1 by fluorescence measurements. The interaction stoichiometry of the TPPS-DMMP system was 1:1, and the association constants were determined to be 16.06M-1 by absorbance measurements and 16.03M-1 by fluorescence measurements. It was concluded that the interaction between TPPS1 and DMMP was stronger than that between TPPS and DMMP.
141 | Kevin M. Clark, Wilfred A. van der Donk, Yi Lu
"Chapter 5 Expressed Protein Ligation for Metalloprotein Design and Engineering"
in "Non-Natural Amino Acids", Methods in Enzymology, Vol. 462, Tom W. Muir, John N. Abelson, eds.; Academic Press; pp. 97-115 (2009) [doi]

Metalloproteins contain highly specialized metal[hyphen (true graphic)]binding sites that are designed to accept specific metal ions to maintain correct function. Although many of the sites have been modified with success, the relative paucity of functional group availability within proteinogenic amino acids can sometimes leave open questions about specific functions of the metal binding ligands. Attaining a more thorough analysis of individual amino acid function within metalloproteins has been realized using expressed protein ligation (EPL). Here we describe our recent efforts using EPL to incorporate nonproteinogenic cysteine and methionine analogues into the type 1 copper site found in Pseudomonas aeruginosa azurin.
140 | Jianhui Zhu, Yongmei Zhao, Yanyan Zhu, Ziyi Wu, Miaoxin Lin, Weijiang He, Yan Wang, Guangju Chen, Lei Dong, Junfeng Zhang, Yi Lu, Zijian Guo
"DNA Cross-Linking Patterns Induced by an Antitumor-Active Trinuclear Platinum Complex and Comparison with Its Dinuclear Analogue"
Chemistry - A European Journal 15, 5245-5253 (2009) [doi]

The center of it all: An antitumor-active trinuclear platinum complex forms unprecedented interstrand cross-linked triadducts with 18-mer DNA duplexes (see figure; complex in yellow with the platinum centers in red) and behaves differently from its dinuclear analogue.The DNA binding and cross-linking modes of a trinuclear platinum complex [Pt3Cl3(hptab)][ClO4]3 (1; hptab=N,N,Nprime,Nprime,Nprimeprime,Nprimeprime-hexakis(2-pyridylmethyl)-1,3,5-tris(aminomethyl)benzene) and its dinuclear analogue [Pt2Cl2(m-tpxa)]Cl2 (2; m-tpxa=N,N,Nprime,Nprime-tetra(2-pyridylmethyl)-m-xylylene diamine) are reported and compared. The adducts of 1 and 2 with 18-mer duplex N1, 5prime-d(GAAGAAGTCACAAAATGT)-3primesdot5prime-d(ACATTTTGTGACTTCTTC)-3prime, have been characterized by means of denaturing polyacrylamide gels, Maxam-Gilbert sequencing, and MALDI-TOF mass spectrometry combined with enzymatic degradation to obtain insights into structural features responsible for the differences in their antitumor activities. The cytotoxic-active complex 1 readily forms various DNA adducts, such as through 1,3- and 1,4-intrastrand cross-links, and in particular, the unique and unprecedented interstrand cross-linked triadducts. In contrast, the cytotoxic-inactive complex 2 preferentially forms 1,4-intrastrand rather than 1,3-intra- and -interstrand cross-links. Digestion of the DNA adducts of 1 shows that the cleavage is completely blocked at one nucleotide before the cross-linked guanine residues on the opposite strand, a feature that appears to be unprecedented in antitumor platinum complexes. In the case of 2, the cleavage bypasses the first platinated guanine site and stops at one nucleotide prior to the second platinated site, confirming that very few 1,3-intrastrand cross-links are formed by 2. These results are supported by molecular-modeling studies of intra- and interstrand cross-links of duplex N1 with 1 and 2. The remarkable differences between 1 and 2 in DNA binding and cross-linking provide mechanistic insights into their different cytotoxicity against the tumor cell lines; these insights are useful for designing future antitumor agents.
139 | B. Ravel, S.C. Slimmer, X. Meng, G.C.L. Wong, Y. Lu
"EXAFS studies of catalytic DNA sensors for mercury contamination of water"
Radiation Physics and Chemistry 78, S75-S79 (2009) [doi]

Monitoring of metallic contaminants in domestic and agricultural water systems requires technology that is fast, flexible, sensitive, and selective. Recently, metal sensors based on catalytic DNA have been demonstrated as a practical monitoring solution. Very little is known, however, about the atomic scale interactions between the DNA-based sensors and the metal contaminant to which the sensor is targeted. Here, we present the results of an X-ray absorption spectroscopy study of a mercury sensor which illustrates the nature of the Hg-DNA interaction.
138 | Yi Lu, Juewen Liu, Debapriya Mazumdar
"Nanoparticles/Dip Stick"
in "Nucleic Acid and Peptide Aptamers: Methods and Protocols", Methods in Molecular Biology, Vol. 535, Gunter Mayer, ed.; pp. 223-239 (2009) [doi]

Aptamers are single-stranded nucleic acids or peptides that can bind target molecules with high affinity and specificity. The conformation of an aptamer usually changes upon binding to its target analyte, and this property has been used in a wide variety of sensing applications, including detections based on fluorescence, electrochemistry, mass, or color change. Because native nucleic acids do not possess signaling moieties required for most detection methods, aptamer sensors usually involve labeling of external signaling groups. Among the many kinds of labels, inorganic nanoparticles are emerging as highly attractive candidates because some of their unique properties. Here, we describe protocols for the preparation of aptamer-linked gold nanoparticles (AuNPs) that undergo fast disassembly into red dispersed nanoparticles upon binding of target analytes. This method has been proven to be generally applicable for colorimetric sensing of a broad range of analytes. The sample protocols have also been successfully applied to quantum dots and magnetic nanoparticles. Finally, to increase the user friendliness of the method, the sensors have been converted into simple dipstick tests using lateral flow devices.
137 | Zidong Wang, Yi Lu
"Functional DNA directed assembly of nanomaterials for biosensing"
J. Mater. Chem. 19, (13) 1788-1798 (2009) [doi] [news]

This review summarizes recent progress in the development of biosensors by integrating functional DNA molecules with different types of nanomaterials, including metallic nanoparticles, semiconductor nanoparticles, magnetic nanoparticles, and carbon nanotubes. On one hand, advances in nanoscale science and technology have generated nanomaterials with unique optical, electrical, magnetic and catalytic properties. On the other hand, recent progress in biology has resulted in functional DNAs, a new class of DNAs that can either bind to a target molecule (known as aptamers) or perform catalytic reactions (known as DNAzymes) with the ability to recognize a broad range of targets from metal ions to organic molecules, proteins and cells specifically. By taking advantage of the strengths in both fields, the physical and chemical properties of nanomaterials have been modulated by the target recognition and catalytic activity of functional DNAs in the presence of a target analyte, resulting in a large number of colorimetric, fluorescent, electrochemical, surface-enhanced Raman scattering and magnetic resonance imaging sensors for the detection of a broad range of analytes with high sensitivity and selectivity.
136 | Zehui Cao, Yi Lu
"New Metallo-DNAzymes: Fundamental Studies of Metal-DNA Interactions and Metal Sensing Applications"
in "Metal Complex-DNA Interactions", Nick Hadjiliadis, Einar Sletten, eds.; Wiley-Blackwell: Oxford, UK; pp. 395-414 (2009) [doi]

This chapter contains sections titled: Introduction Metal Ions as Important Cofactors of DNAzymes Selection of DNAzymes Using in vitro Evolution Understanding Nucleic Acid Enzyme-Metal Ion Interactions DNAzymes as Metal Ion Sensors Summary Acknowledgements References
135 | Yingfu Li, Yi Lu
"Functional Nucleic Acids for Sensing and Other Analytical Applications"
Integrated Analytical Systems, Springer: New York, NY; (2009) [archived PDF] [doi]
134 | Juewen Liu, Yi Lu
"Colorimetric and Fluorescent Biosensors Based on Directed Assembly of Nanomaterials with Functional DNA"
in "Functional Nucleic Acids for Analytical Applications", Integrated Analytical Systems, Yingfu Li, Yi Lu, eds.; Springer: New York, NY; pp. 155-178 (2009) [doi]

This chapter reviews recent progress in the interface between functional nucleic acids and nanoscale science and technology, and its analytical applications. In particular, the use of metallic nanoparticles as the color reporting groups for the action (binding, catalysis, or both) of aptamers, DNAzymes, and aptazymes is described in detail. Because metallic nanoparticles possess high extinction coefficients and distance-dependent optical properties, they allow highly sensitive detections with minimal consumption of materials. The combination of quantum dots (QDs) with functional nucleic acids as fluorescent sensors is also described. The chapter starts with the design of colorimetric and fluorescent sensors responsive to single analytes, followed by sensors responsive to multiple analytes with controllable cooperativity and multiplex detection using both colorimetric and fluorescent signals in one pot, and ends by transferring solution-based detections into litmus paper type of tests, making them generally applicable and usable for a wide range of on-site and real-time analytical applications such as household tests, environmental monitoring, and clinical diagnostics.
133 | Andrea K. Brown, Juewen Liu, Ying He, Yi Lu
"Biochemical Characterization of a Uranyl Ion-Specific DNAzyme"
ChemBioChem 10, 486-492 (2009) [doi]

Uranyl ion-specific DNAzyme: A DNAzyme (lower strand) cleaves the substrate (upper strand) in the presence of the uranyl ion. The enzyme folds into a bulged three-way-junction structure with catalytically important nucleotides residing in the bulge. A highly conserved GsdotA mismatch is also crucial for the enzyme's activity.The biochemical characterization of a DNAzyme that is highly specific for uranyl (UO22+) ions is described. Sequence alignment, enzyme truncation, and mutation studies have resulted in a conserved sequence that folds into a bulged stem-loop structure. Interestingly, a GsdotA pair next to the scissile site is important for the uranyl ion-specific DNAzyme; this is reminiscent of the GsdotT wobble base pair adjacent to the cleavage site that is crucial for the PbII-specific 8-17 DNAzyme activity. Therefore wobble pairs might be important for formation of metal-specific metal-binding sites in DNAzymes. The DNAzyme binds the uranyl ion with a dissociation constant of 469 nM, which is among the strongest metal-binding affinities in nucleic acid enzymes reported to date. This explains why a catalytic beacon fluorescent sensor based on this enzyme has a detection limit (45 pM) that rivals the most-sensitive analytical instrument. It also has over 1?000?000-fold specificity for the uranyl ion over other metal ions. The DNAzyme can carry out multiple turnover reactions that follow the Michaelis-Menten equation, with a kcat of 1.46 min-1 and a KM of 463 nM, similar to that of the 8-17 DNAzyme. The pH profile shows a bell-shaped curve that reaches a maximum at pH 5.5, at which the in vitro selection was carried out; this suggests that in vitro selection can be performed to obtain DNAzymes with optimal performance under specific conditions under which practical applications are required. These findings enrich our fundamental understanding of metal-binding sites in nucleic acids and allow the design of sensors with better performance.
132 | Jang-Ung Park, Jung Heon Lee, Ungyu Paik, Yi Lu, John A. Rogers
"Nanoscale Patterns of Oligonucleotides Formed by Electrohydrodynamic Jet Printing with Applications in Biosensing and Nanomaterials Assembly"
Nano Letters 8, 4210-4216 (2008) [PDF] [doi]

The widespread use of DNA in microarrays for applications in biotechnology, combined with its promise in programmed nanomaterials assembly, unusual electronic devices, and other areas has created interest in methods for patterning DNA with high spatial resolution. Techniques based on thermal or piezoelectric inkjet printing are attractive due to their noncontacting nature and their compatibility with diverse materials and substrate types; their modest resolution (i.e., 10−20 μm) represents a major limitation for certain systems. Here we demonstrate the use of an operationally similar printing approach that exploits electrohydrodynamic forces, rather than thermal or acoustic energy, to eject DNA inks through fine nozzles, in a controlled fashion. This DNA printer is capable of resolution approaching 100 nm. A range of experiments on patterns of DNA formed with this printer demonstrates its key features. Example applications in DNA-directed nanoparticle assembly and DNA aptamer-based biosensing illustrate two representative uses of the patterns that can be formed.
131 | Zidong Wang, Jung Heon Lee, Yi Lu
"Highly sensitive "turn-on" fluorescent sensor for Hg2+ in aqueous solution based on structure-switching DNA"
Chemical Communications 2008, 6005-6007 (2008) [doi]

fluorescent sensor for mercury was demonstrated based on structure-switching DNA with a low detection limit of 3.2 nM and high selectivity.
130 | Masha G. Savelieff, Tiffany D. Wilson, Youssef Elias, Mark J. Nilges, Dewain K. Garner, Yi Lu
"Experimental evidence for a link among cupredoxins: Red, blue, and purple copper transformations in nitrous oxide reductase"
Proceedings of the National Academy of Sciences 105, 7919-7924 (2008) [PDF] [doi] [news]

The cupredoxin fold is an important motif in numerous proteins that are central to several critical cellular processes ranging from aerobic and anaerobic respiration to catalysis and iron homeostasis. Three types of copper sites have been found to date within cupredoxin folds: blue type 1 (T1) copper, red type 2 (T2) copper, and purple CuA. Although as much as 90% sequence difference has been observed among some members of this superfamily of proteins that span several kingdoms, sequence alignment and phylogenic trees strongly suggest an evolutionary link and common ancestry. However, experimental evidence for such a link has been lacking. We report herein the observation of pH-dependent transformation between blue T1 copper, red T2 copper, and the native purple CuA centers of nitrous oxide reductase (N2OR) from Paracoccus denitrificans. The blue and red copper centers form initially before they are transformed into purple CuA center. This transformation process is pH-dependent, with lower pH resulting in fewer trapped T1 and T2 coppers and faster transition to purple CuA. These observations suggest that the purple CuA site contains the essential elements of T1 and T2 copper centers and that the CuA center is preferentially formed at low pH. Therefore, this work provides an underlying link between the various cupredoxin copper sites and possible experimental evidence in vitro for the evolutionary relationship between the cupredoxin proteins. The findings also lend physiological relevance to cupredoxin site biosynthesis.
129 | Jung Heon Lee, Zidong Wang, Juewen Liu, Yi Lu
"Highly Sensitive and Selective Colorimetric Sensors for Uranyl (UO22+): Development and Comparison of Labeled and Label-Free DNAzyme-Gold Nanoparticle Systems"
Journal of the American Chemical Society 130, 14217-14226 (2008) [PDF] [archived PDF] [doi]

Colorimetric uranium sensors based on uranyl (UO22+) specific DNAzyme and gold nanoparticles (AuNP) have been developed and demonstrated using both labeled and label-free methods. In the labeled method, a uranyl-specific DNAzyme was attached to AuNP, forming purple aggregates. The presence of uranyl induced disassembly of the DNAzyme functionalized AuNP aggregates, resulting in red individual AuNPs. Once assembled, such a “turn-on” sensor is highly stable, works in a single step at room temperature, and has a detection limit of 50 nM after 30 min of reaction time. The label-free method, on the other hand, utilizes the different adsorption properties of single-stranded and double-stranded DNA on AuNPs, which affects the stability of AuNPs in the presence of NaCl. The presence of uranyl resulted in cleavage of substrate by DNAzyme, releasing a single stranded DNA that can be adsorbed on AuNPs and protect them from aggregation. Taking advantage of this phenomenon, a “turn-off” sensor was developed, which is easy to control through reaction quenching and has 1 nM detection limit after 6 min of reaction at room temperature. Both sensors have excellent selectivity over other metal ions and have detection limits below the maximum contamination level of 130 nM for UO22+ in drinking water defined by the U.S. Environmental Protection Agency (EPA). This study represents the first direct systematic comparison of these two types of sensor methods using the same DNAzyme and AuNPs, making it possible to reveal advantages, disadvantages, versatility, limitations, and potential applications of each method. The results obtained not only allow practical sensing application for uranyl but also serve as a guide for choosing different methods for designing colorimetric sensors for other targets.
128 | Zidong Wang, Jung Heon Lee, Yi Lu
"Label-Free Colorimetric Detection of Lead Ions with a Nanomolar Detection Limit and Tunable Dynamic Range by using Gold Nanoparticles and DNAzyme"
Advanced Materials 20, 3263-3267 (2008) [doi]

In the presence of Pb2+, a cleaved enzyme-substrate complex releases ssDNA that adsorbs onto and stabilizes gold nanoparticles (AuNPs) against salt-induced aggregation. In the absence of Pb2+, the uncleaved complex can not stabilize the AuNPs, resulting in purple-blue AuNP aggregates (see figure). The sensor has a low detection limit of 3?nM, a high selectivity, and a tunable dynamic range.
127 | Hee-Kyung Kim, Jing Li, Nandini Nagraj, Yi Lu
"Probing Metal Binding in the 8-17?DNAzyme by TbIII Luminescence Spectroscopy"
Chemistry - A European Journal 14, 8696-8703 (2008) [doi]

Metal-dependent cleavage activities of the 8-17 DNAzyme were found to be inhibited by TbIII ions, and the apparent inhibition constant in the presence of 100?muM of ZnII was measured to be 3.3\textpm0.3?muM. The apparent inhibition constants increased linearly with increasing ZnII concentration, and the inhibition effect could be fully rescued with addition of active metal ions, indicating that TbIII is a competitive inhibitor and that the effect is completely reversible. The sensitized TbIII luminescence at 543?nm was dramatically enhanced when TbIII was added to the DNAzyme-substrate complex. With an inactive DNAzyme in which the GT wobble pair was replaced with a GC Watson-Crick base pair, the luminescence enhancement was slightly decreased. In addition, when the DNAzyme strand was replaced with a complete complementary strand to the substrate, no significant luminescence enhancement was observed. These observations suggest that TbIII may bind to an unpaired region of the DNAzyme, with the GT wobble pair playing a role. Luminescence lifetime measurements in D2O and H2O suggested that TbIII bound to DNAzyme is coordinated by 6.7\textpm0.2 water molecules and two or three functional groups from the DNAzyme. Divalent metal ions competed for the TbIII binding site(s) in the order CoII>ZnII>MnII>PbII>CaII≈MgII. This order closely follows the order of DNAzyme activity, with the exception of PbII. These results indicate that PbII, the most active metal ion, competes for TbIII binding differently from other metal ions such as ZnII, suggesting that PbII may bind to a different site from that for the other metal ions including ZnII and TbIII.
126 | Yi Lu, Juewen Liu
"Catalyst-functionalized nanomaterials"
Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 1, 35-46 (2008) [doi]

With rapid development in both nanotechnology and biotechnology, it is now possible to combine these two exciting fields to modulate the physical properties of nanomaterials with the molecular recognition and catalytic functional properties of biomolecules. Such research efforts have resulted in a larger number of sensors that can detect a broad range of analytes ranging from metal ions, small molecules, and nucleic acids down to proteins. These sensors will find important applications in nanomedicine. In this article, the design of sensors with four classes of nanomaterials (metallic, semiconductor, magnetic, and carbon nanotube nanoparticles) is reviewed. Metallic nanoparticles possess distance-dependent optical properties and are useful for designing colorimetric sensors. Semiconductor nanoparticles or quantum dots (QDs) appear to be superior alternatives to traditional organic fluorophores in many aspects, such as broad excitation range, narrow emission peaks, and high photo stability. QD sensors based on either energy transfer or charge transfer are summarized. Furthermore, magnetic nanoparticles are shown to be useful as smart magnetic resonance imaging (MRI) contrast agents. Finally, some carbon nanotubes show near-IR emission properties, and thus, are potentially useful for in vivo sensing. Sensors based on either tuning the emission intensity or wavelength are discussed. Copyright \textcopyright 2008 John Wiley & Sons, Inc.For further resources related to this article, please visit the .
125 | Debapriya Mazumdar, Juewen Liu, Yi Lu
"Functional Nucleic Acid-Directed Assembly of Nanomaterials and their Applications as Colorimetric and Fluorescent Sensors for Trace Contaminants in Water"
in "Nanotechnology Applications for Clean Water", Nora Savage, Mamadou Diallo, Jeremiah Duncan, Anita Street, Richard Sustich, eds.; pp. 427-446 (2008)
123 | Margaret H. S. Shyr, Daryl P. Wernette, Pierre Wiltzius, Yi Lu, Paul V. Braun
"DNA and DNAzyme-Mediated 2D Colloidal Assembly"
Journal of the American Chemical Society 130, 8234-8240 (2008) [PDF] [doi]

DNA-mediated interactions present a significant opportunity for controlling colloidal self-assembly. Using microcontact printing to achieve spatial control of DNA-surface patterning and DNA-functionalized polystyrene colloids, we report that DNA hybridization can be utilized for sequence-specific reversible self-assembly of well-ordered 2D colloidal arrays. Two essential indicators of DNA-hybridization mediated assembly were confirmed: thermal reversibility and sequence specificity. The arrays melted at 50 °C and reassembled when introduced to fresh colloid suspension, and sequence specificity with <1% nonspecific binding was confirmed using fluorescent polystyrene colloids. The real-time assembly of the colloids onto the periodically patterned substrate was monitored by simple laser diffraction to obtain assembly kinetics. Maximum surface coverage of DNA-mediated assembly was determined to be 0.593 for DNA-functionalized 100 nm polystyrene colloids, and 90% of the assembly was complete after 6.25 h of hybridization in 50 mM NaCl Tris buffer. We also demonstrate that DNAzymes, catalytic DNA molecules, can be incorporated into the design, and in the presence of 10 µM Pb2+, the hybridization-induced array assembly can be disrupted via DNAzyme activity.
122 | Tulika S. Dalavoy, Daryl P. Wernette, Maojun Gong, Jonathan V. Sweedler, Yi Lu, Bruce R. Flachsbart, Mark A. Shannon, Paul W. Bohn, Donald M. Cropek
"Immobilization of DNAzyme catalytic beacons on PMMA for Pb2+ detection"
Lab on a Chip 8, 786-793 (2008) [doi]

Due to the numerous toxicological effects of lead, its presence in the environment needs to be effectively monitored. Incorporating a biosensing element within a microfluidic platform enables rapid and reliable determinations of lead at trace levels. A microchip-based lead sensor is described here that employs a lead-specific DNAzyme (also called catalytic DNA or deoxyribozyme) as a recognition element that cleaves its complementary substrate DNA strand only in the presence of cationic lead (Pb2+). Fluorescent tags on the DNAzyme translate the cleavage events to measurable, optical signals proportional to Pb2+ concentration. The DNAzyme responds sensitively and selectively to Pb2+, and immobilizing DNAzyme in the sensor permits both sensor regeneration and localization of the detection zone. Here, the DNAzyme has been immobilized on a PMMA surface using the highly specific biotin-streptavidin interaction. The strategy includes using streptavidin physisorbed on a PMMA surface to immobilize DNAzyme both on planar PMMA and on the walls of a PMMA microfluidic device. The immobilized DNAzyme retains its Pb2+ detection activity in the microfluidic device and can be regenerated and reused. The DNAzyme shows no response to other common metal cations and the presence of these contaminants does not interfere with the lead-induced fluorescence signal. While prior work has shown lead-specific catalytic DNA can be used in its solubilized form and while attached to gold substrates to quantitate Pb2+ in solution, this is the first use of the DNAzyme immobilized within a microfluidic platform for real time Pb2+ detection.
121 | Xiangjin Xie, Serge I. Gorelsky, Ritimukta Sarangi, Dewain K. Garner, Hee Jung Hwang, Keith O. Hodgson, Britt Hedman, Yi Lu, Edward I. Solomon
"Perturbations to the Geometric and Electronic Structure of the CuA Site: Factors that Influence Delocalization and Their Contributions to Electron Transfer"
Journal of the American Chemical Society 130, 5194-5205 (2008) [PDF] [doi]

Using a combination of electronic spectroscopies and DFT calculations, the effect of pH perturbation on the geometric and electronic structure of the CuA site has been defined. Descriptions are developed for high pH (pH = 7) and low pH (pH = 4) forms of CuA azurin and its H120A mutant which address the discrepancies concerning the extent of delocalization indicated by multifrequency EPR and ENDOR data (J. Am. Chem. Soc. 2005, 127, 7274; Biophys. J. 2002, 82, 2758). Our resonance Raman and MCD spectra demonstrate that the low pH and H120A mutant forms are essentially identical and are the perturbed forms of the completely delocalized high pH CuA site. However, in going from high pH to low pH, a seven-line hyperfine coupling pattern associated with complete delocalization of the electron (S = 1/2) over two Cu coppers (ICu = 3/2) changes into a four-line pattern reflecting apparent localization. DFT calculations show that the unpaired electron is delocalized in the low pH form and reveal that its four-line hyperfine pattern results from the large EPR spectral effects of ∼1% 4s orbital contribution of one Cu to the ground-state spin wave function upon protonative loss of its His ligand. The contribution of the Cu−Cu interaction to electron delocalization in this low symmetry protein site is evaluated, and the possible functional significance of the pH-dependent transition in regulating proton-coupled electron transfer in cytochrome c oxidase is discussed.
120 | Yi Lu, Dewain K. Garner, Jun-long Zhang, Tadhg P. Begley
"Artificial Metalloproteins: Design and Engineering"
in "Wiley Encyclopedia of Chemical Biology", John Wiley & Sons, Inc.; (2008) [archived PDF] [doi]

Engineering of artificial metalloproteins is an expanding field with potential impacts in many areas from fundamental understanding of protein structure and function to industrial production of specialty chemicals such as chiral drug intermediates. Incorporation of unnatural amino acids and non-native metal cofactors into proteins is an emerging field in the area of protein design, as it offers the tantalizing prospect of introducing new functionality and provides exquisite probes for and fine-tuning of native protein properties. Although it is a relatively young field, engineering of non-native metalloproteins boasts a myriad of techniques for design, construction, and/or expression of the desired artificial protein. Additionally, many groundbreaking studies exist in this field that have enhanced basic scientific understanding of protein function or generated promising artificial enzymes. Discussion of the techniques prominent for incorporation of unnatural amino acids and non-native cofactors is followed by some of the most interesting applications of these techniques reported to date.
119 | Jun-Long Zhang, Dewain K. Garner, Lei Liang, Qian Chen, Yi Lu
"Protein scaffold of a designed metalloenzyme enhances the chemoselectivity in sulfoxidation of thioanisole"
Chemical Communications , 1665-1667 (2008) [doi]

We demonstrate that incorporation of MnSalen into a protein scaffold enhances the chemoselectivity in sulfoxidation of thioanisole and find that both the polarity and hydrogen bonding of the protein scaffold play an important role in tuning the chemoselectivity.
118 | Ritimukta Sarangi, Serge I. Gorelsky, Lipika Basumallick, Hee Jung Hwang, Russell C. Pratt, T. Daniel P. Stack, Yi Lu, Keith O. Hodgson, Britt Hedman, Edward I. Solomon
"Spectroscopic and Density Functional Theory Studies of the Blue−Copper Site in M121SeM and C112SeC Azurin:  Cu−Se Versus Cu−S Bonding"
Journal of the American Chemical Society 130, 3866-3877 (2008) [PDF] [doi]

S K-edge X-ray absorption, UV−vis absorption, magnetic circular dichroism (MCD), and resonance Raman spectroscopies are used to investigate the electronic structure differences among WT, M121SeM, and C112SeC Pseudomonas aeruginosa (P.a) azurin. A comparison of S K-edge XAS of WT and M121SeM azurin and a CuII−thioether model complex shows that the 38% S character in the ground state wave function of the blue−copper (BC) sites solely reflects the Cu−SCys bond. Resonance Raman (rR) data on WT and C112SeC azurin give direct evidence for the kinematic coupling between the Cu−SCys stretch and the cysteine deformation modes in WT azurin, which leads to multiple features in the rR spectrum of the BC site. The UV−vis absorption and MCD data on WT, M121SeM, and C112SeC give very similar C0/D0 ratios, indicating that the C-term MCD intensity mechanism involves Cu-centered spin−orbit coupling (SOC). The spectroscopic data combined with density functional theory (DFT) calculations indicate that SCys and SeCys have similar covalent interactions with Cu at their respective bond lengths of 2.1 and 2.3 Å. This reflects the similar electronegativites of S and Se in the thiolate/selenolate ligand fragment and explains the strong spectroscopic similarities between WT and C112SeC azurin.
117 | Masha G. Savelieff, Yi Lu
"pH dependent copper binding properties of a CuA azurin variant with both bridging cysteines replaced with serines"
Inorganica Chimica Acta 361, 1087-1094 (2008) [doi]

A double mutant of CuA azurin was prepared in which both bridging cysteine thiolate ligands of the binuclear CuA center were replaced by serine. The copper binding properties of this protein were investigated, and shown to be pH dependent. At lower pH (5.20.1), the protein binds one copper per protein molecule as demonstrated by electrospray ionization mass spectrometry. Copper titrations resulted in electronic absorptions at 730nm (peak) and ca. 330nm (shoulder) in the UV-Vis spectrum. EPR data show a four line pattern with hyperfine A[short parallel]=150G and g[short parallel] and g[perpendicular] values 2.32 and 2.03, characteristic of a type II (T2) copper. Superhyperfines to two nitrogen atoms were also observed. At higher pH (8.50.1), the protein binds upto two copper atoms per protein molecule, and copper titrations exhibit a blue transition at 595nm in the UV-Vis spectrum. The EPR data are consistent with two monomeric sites very similar to one another having hyperfines A[short parallel]=182 and 150G, g[short parallel]=2.24 and 2.22 and a similar g[perpendicular] value of 2.01. These results indicate that both bridging cysteines play a critical role in the CuA center, and replacing them with serines is not enough to maintain the symmetrical diamond core structure or the characteristic electronic and functional properties of the CuA center.
116 | Daryl P. Wernette, Juewen Liu, Paul W. Bohn, Yi Lu
"Functional DNA-Based Nanoscale Materials and Devices for Sensing Trace Contaminants in Water"
MRS Bulletin 33, 34-41 (2008) [PDF]
115 | Mehmet Veysel Yigit, Debapriya Mazumdar, Yi Lu
"MRI Detection of Thrombin with Aptamer Functionalized Superparamagnetic Iron Oxide Nanoparticles"
Bioconjugate Chemistry 19, 412-417 (2008) [PDF] [doi] [news]

The detection of the human α-thrombin protein by MRI is reported by designing a thrombin targeted MRI contrast agent. The contrast agent is composed of thrombin aptamer functionalized superparamagnetic iron oxide nanoparticles. The detection of thrombin is based on the increase in the size of nanoparticle assembly, which leads to a change in brightness of the image. A detectable change in MR signal is observed with 25 nM thrombin in human serum.
114 | Nathan A. Sieracki, Hee Jung Hwang, Michelle K. Lee, Dewain K. Garner, Yi Lu
"A temperature independent pH (TIP) buffer for biomedical biophysical applications at low temperatures"
Chemical Communications , 823-825 (2008) [doi] [news]

A temperature independent pH buffer has been developed from a combination of buffers of opposite-sign temperature coefficients, and utility in low temperature spectroscopy and storage of pH sensitive compounds is demonstrated.
113 | Lauren A. Denofrio, Brandy Russell, David Lopatto, Yi Lu
"Linking Student Interests to Science Curricula"
Science 318, 1872-1873 (2007) [PDF] [doi] [news]
112 | Hee-Kyung Kim, Ivan Rasnik, Juewen Liu, Taekjip Ha, Yi Lu
"Dissecting metal ion-dependent folding and catalysis of a single DNAzyme"
Nat Chem Biol 3, 763-768 (2007) [doi] [news]
111 | Juewen Liu, Yi Lu
"Colorimetric Cu2+ detection with a ligation DNAzyme and nanoparticles"
Chemical Communications , 4872-4874 (2007) [doi] [news]

Using a Cu2+-dependent DNA ligation DNAzyme, a colorimetric sensor for Cu2+ has been developed based on directed assembly of DNA-functionalized gold nanoparticles by the ligation product, and such ligation DNAzyme-based sensors are intrinsically more sensitive than cleavage DNAzyme systems due to the lack of background.
110 | Jung Heon Lee, Daryl P. Wernette, Mehmet V. Yigit, Juewen Liu, Zidong Wang, Yi Lu
"Site-Specific Control of Distances between Gold Nanoparticles Using Phosphorothioate Anchors on DNA and a Short Bifunctional Molecular Fastener"
Angewandte Chemie International Edition 46, 9006-9010 (2007) [doi]

Going for double gold: Precise control of the positions of and distances between gold nanoparticles (AuNP; gold spheres in picture) is achieved by linking the AuNPs to phosphorothioate-modified DNA (green and red helices) with a bifunctional fastener. The distance between AuNPs is controlled simply by changing the position of the modifications on the DNA template.
109 | Juewen Liu, Yi Lu
"Rational Design of "Turn-On" Allosteric DNAzyme Catalytic Beacons for Aqueous Mercury Ions with Ultrahigh Sensitivity and Selectivity"
Angewandte Chemie International Edition 46, 7587-7590 (2007) [doi]

The mercury is rising: A DNAzyme with several thymine residues located close to the catalytic site is rendered inactive. However, thymine-Hg2+-thymine interactions transform the DNAzyme into an active enzyme. When fluorophore/quencher pairs are attached, the DNAzyme can be converted into a highly sensitive and selective mercury sensor with a detection limit of 2.4?nM and a greater than 100?000-fold selectivity over other metal ions.
108 | Mehmet Veysel Yigit, Debapriya Mazumdar, Hee-Kyung Kim, Jung Heon Lee, Boris Odintsov, Yi Lu
"Smart "Turn-on" Magnetic Resonance Contrast Agents Based on Aptamer-Functionalized Superparamagnetic Iron Oxide Nanoparticles"
ChemBioChem 8, 1675-1678 (2007) [doi]

Smart agents. A contrast agent was designed by combining target specific nucleic-acid aptamers against adenosine with superparamagnetic iron oxide nanoparticles. Target-induced disassembly of clustered nanoparticles in the presence of adenosine led to an increase in T2, which was seen as an increase in the brightness of the magnetic resonance image (see figure).
107 | Juewen Liu, Yi Lu
"A DNAzyme Catalytic Beacon Sensor for Paramagnetic Cu2+ Ions in Aqueous Solution with High Sensitivity and Selectivity"
Journal of the American Chemical Society 129, 9838-9839 (2007) [PDF] [doi]

Copper is a key metal ion both in environment monitoring and in biology, and exposure to high concentration of copper can cause adverse health effects. Although significant progresses have been made in designing fluorescent sensors for diamagnetic metal ions, few effective Cu2+ sensors are known because of the paramagnetic nature of the metal ion. We herein report a highly sensitive and selective “turn-on” fluorescent Cu2+ sensor based on an in vitro selected DNAzyme (also known as catalytic DNA or deoxyribozyme). The substrate strand of the DNAzyme was labeled with a fluorophore on the 3‘-end and a quencher on the 5‘-end, and the enzyme strand was labeled with a second quencher on the 5‘-end. Initially, the fluorescence was quenched. Addition of Cu2+ induced oxidative cleavage of the substrate, and the fluorescence intensity increased by 13-fold. The sensor has a detection limit of 35 nM and a dynamic range up to 20 μM. The sensor selectivity is more than 2000-fold for Cu2+ over Fe2+ and UO22+ and more than 10 000-fold over any other metal ions. The DNAzyme catalytic beacon method demonstrated here can be applied to designing turn-on fluorescent sensors for other paramagnetic metal ions.
106 | Daryl P. Wernette, Carolyn Mead, Paul W. Bohn, Yi Lu
"Surface Immobilization of Catalytic Beacons Based on Ratiometric Fluorescent DNAzyme Sensors:  A Systematic Study"
Langmuir 23, 9513-9521 (2007) [PDF] [doi]

DNAzyme-based catalytic beacons have the potential for sensing a large number of relevant analytes. Thus, a systematic investigation of factors affecting their performance when immobilized into gold-coated nanocapillary array membranes (NCAMs) was undertaken. Enzyme immobilization times were varied to determine that as little as 15 min was sufficient for ratiometric detection of Pb2+-specific activity, while immobilization density saturated after 1.5 h. Immobilization of the DNAzymes into NCAMs with 600 nm pore size resulted in higher immobilization efficiency and higher enzymatic activity than that with 200 nm pore size. A poly-T linker length between the tethering thiol and first oligonucleotide, used to extend the DNAzyme above the backfilling mercaptohexanol (MCH) monolayer, had no effect on DNAzyme activity. The backfilling method of immobilization, involving backfilling followed by hybridization, was found most effective for DNAzyme activity compared to immobilization of hybridized DNAzyme complex (a 67% loss of activity) or concurrent enzyme and MCH immobilization (75% loss of activity). The backfilling MCH monolayer provided ∼3.5 times increase in activity compared to DNAzyme assembled without MCH, and was over 5 times more active than shorter and longer backfilling molecules tested. The immobilized DNAzyme retained its optimized performance at 50 mM NaCl. Finally, the generalized immobilization and ratiometric procedure was employed for a uranyl-specific DNAzyme with 25 ± 15 times increase in ratio observed. These findings form a firm basis on which practical applications of catalytic beacons can be realized, including sensors for both Pb2+ and UO22+ ions.
105 | Juewen Liu, Yi Lu
"Non-Base Pairing DNA Provides a New Dimension for Controlling Aptamer-Linked Nanoparticles and Sensors"
Journal of the American Chemical Society 129, 8634-8643 (2007) [PDF] [doi]

DNA aptamers have been recently applied as simple and fast colorimetric sensors for a wide range of molecules. A unique feature of these systems is the presence of non-base pairing oligonucleotides in both DNA aptamers and spacers on DNA-functionalized nanoparticles. We report here a systematic investigation on an adenosine aptamer-linked gold nanoparticle system. When the aptamer overhang and the spacer were aligned on the same side, adenosine-responsive disassembly was inhibited. This inhibition effect increased with the length of the spacer, and fully inhibited activity was observed with the spacer containing more than three nucleotides. In contrast to a linear relationship between the spacer length and melting temperature in double-stranded DNA systems without overhangs, the aptamer system displayed a nonlinear relationship, with the melting temperature decreasing exponentially with spacer length. Control experiments suggested that this inhibition effect was due to thermodynamic factors rather than kinetic traps. A comparison with aptamer beacon systems indicated that nanoparticles may play an important role in this inhibition effect, and no specific interactions between the aptamer overhang and spacer were detected. The identity of nucleotides in the spacer did not affect the conclusions. Furthermore, the rate of disassembly or color change was slower at lower temperature or higher ionic strength, but was little affected by pH from 5.2 to 9.2. Therefore, non-base pairing DNA provided another dimension for controlling DNA-linked nanoparticles in addition to pH, temperature, or ionic strength, and this knowledge has resulted in the most optimal construct for sensing applications.
104 | Hee-Kyung Kim, Juewen Liu, Jing Li, Nandini Nagraj, Mingxi Li, Caroline M.-B. Pavot, Yi Lu
"Metal-Dependent Global Folding and Activity of the 8-17 DNAzyme Studied by Fluorescence Resonance Energy Transfer"
Journal of the American Chemical Society 129, 6896-6902 (2007) [PDF] [doi]

The 8-17 DNAzyme is a DNA metalloenzyme catalyzing RNA transesterification in the presence of divalent metal ions, with activity following the order Pb2+ ≫ Zn2+ ≫ Mg2+. Since the DNAzyme has been used as a metal ion sensor, its metal-induced global folding was studied by fluorescence resonance energy transfer (FRET) by labeling the three stems of the DNAzyme with the Cy3/Cy5 FRET pair two stems at a time in order to gain deeper insight into the role of different metal ions in its structure and function. FRET results indicated that, in the presence of Zn2+ and Mg2+, the DNAzyme folds into a compact structure, stem III approaching a configuration defined by stems I and II without changing the angle between stems I and II. Correlations between metal-induced folding and activity were also studied. For Zn2+ and Mg2+, the metal ion with higher affinity for the DNAzyme in global folding (Kd(Zn) = 52.6 μM and Kd(Mg) = 1.36 mM) also displays higher affinity in activity (Kd(Zn) = 1.15 mM and Kd(Mg) = 53 mM) under the same conditions. Global folding was saturated at much lower concentrations of Zn2+ and Mg2+ than the cleavage activities, indicating the global folding of the DNAzyme occurs before the cleavage activity for those metal ions. Surprisingly, no Pb2+-dependent global folding was observed. These results suggest that for Pb2+ global folding of the DNAzyme may not be a necessary step in its function, which may contribute to the DNAzyme having the highest activity in the presence of Pb2+.
103 | Juewen Liu, Jung Heon Lee, Yi Lu
"Quantum Dot Encoding of Aptamer-Linked Nanostructures for One-Pot Simultaneous Detection of Multiple Analytes"
Analytical Chemistry 79, 4120-4125 (2007) [PDF] [doi] [news]

One major challenge in analytical chemistry is multiplex sensing of a number of analytes with each analyte displaying a different signal. To meet such a challenge, quantum dots that emit at 525 and 585 nm are used to encode aptamer-linked nanostructures sensitive to adenosine and cocaine, respectively. In addition to quantum dots, the nanostructures also contain gold nanoparticles that serve as quenchers. Addition of target analytes disassembles the nanostructures and results in increased emission from quantum dots. Simultaneous colorimetric and fluorescent detection and quantification of both molecules in one pot is demonstrated.
102 | Juewen Liu, Andrea K. Brown, Xiangli Meng, Donald M. Cropek, Jonathan D. Istok, David B. Watson, Yi Lu
"A catalytic beacon sensor for uranium with parts-per-trillion sensitivity and millionfold selectivity"
Proceedings of the National Academy of Sciences 104, 2056-2061 (2007) [PDF] [doi] [news]

Here, we report a catalytic beacon sensor for uranyl (UO2 2+) based on an in vitro-selected UO2 2+-specific DNAzyme. The sensor consists of a DNA enzyme strand with a 3′ quencher and a DNA substrate with a ribonucleotide adenosine (rA) in the middle and a fluorophore and a quencher at the 5′ and 3′ ends, respectively. The presence of UO2 2+ causes catalytic cleavage of the DNA substrate strand at the rA position and release of the fluorophore and thus dramatic increase of fluorescence intensity. The sensor has a detection limit of 11 parts per trillion (45 pM), a dynamic range up to 400 nM, and selectivity of >1-million-fold over other metal ions. The most interfering metal ion, Th(IV), interacts with the fluorescein fluorophore, causing slightly enhanced fluorescence intensity, with an apparent dissociation constant of ≈230 μM. This sensor rivals the most sensitive analytical instruments for uranium detection, and its application in detecting uranium in contaminated soil samples is also demonstrated. This work shows that simple, cost-effective, and portable metal sensors can be obtained with similar sensitivity and selectivity as much more expensive and sophisticated analytical instruments. Such a sensor will play an important role in environmental remediation of radionuclides such as uranium.
101 | Ole Farver, Hee Jung Hwang, Yi Lu, Israel Pecht
"Reorganization Energy of the CuA Center in Purple Azurin:  Impact of the Mixed Valence-to-Trapped Valence State Transition†"
The Journal of Physical Chemistry B 111, 6690-6694 (2007) [PDF] [doi]

Mixed valence (MV) coordination compounds play important roles in redox reactions in chemistry and biology. Details of the contribution of a mixed valence state to protein electron transfer (ET) reactivity such as reorganization energy, however, have not been experimentally defined. Herein we report measurements of reorganization energies of a binuclear CuA center engineered into Pseudomonas aeruginosa azurin that exhibits a reversible transition between a totally delocalized MV state at pH 8.0 and a trapped valence (TV) state at pH 4.0. The reorganization energy of a His120Ala variant of CuA azurin that displays a TV state at both the above pH values has also been determined. We found that the MV-to-TV state transition increases the reorganization energy by 0.18 eV, providing evidence that the MV state of the CuA center has lower reorganization energy than its TV counterpart. We have also shown that lowering the pH from 8.0 to 4.0 results in a similar (∼0.4 eV) decrease in reorganization energy for both blue (type 1) and purple (CuA) azurins, even though the reorganization energies of the two different copper centers are different at a given pH. These results suggest that the MV state plays only a secondary role in modulation of the ET reactivity via the reorganization energy, as compared to that of the driving force.
100 | Yi Lu, Juewen Liu
"Smart Nanomaterials Inspired by Biology: Dynamic Assembly of Error-Free Nanomaterials in Response to Multiple Chemical and Biological Stimuli"
Accounts of Chemical Research 40, 315-323 (2007) [PDF] [doi] [news]

Three-dimensional functional nanoscale assembly requires not only self-assembly of individual nanomaterials responsive to external stimuli, such as temperature, light, and concentrations, but also directed assembly of many different nanomaterials in one-pot responsive to multiple internal stimuli signaling the needs for such materials at a specific location and a particular time. The use of functional DNA (DNAzymes, aptamers, and aptazymes) to meet these challenges is reviewed. In addition, a biology-inspired proof-reading and error correction method is introduced to cope with errors in nanomaterials assembly.
99 | Yi Lu, Thomas D. Pfister
"The Ubiquitous Roles of Cytochrome P450 Proteins"
in "Metal Ions in Life Sciences", Vol. 3, Astrid Sigel, Helmut Sigel, Roland K. O. Sigel, eds.; John Wiley & Sons, Ltd: Chichester, UK; pp. 267-284 (2007) [doi]

This chapter contains sections titled: Overview of Cytochrome P450 Active Site Structure Secondary Coordination Sphere on the Proximal Side Secondary Coordination Sphere on the Distal Side Summary and Outlook Acknowledgments Abbreviations References
98 | Thomas Pfister, Amir Mirarefi, Alan Gengenbach, Xuan Zhao, Connor Danstrom, Nicole Conatser, Yi-Gui Gao, Howard Robinson, Charles Zukoski, Andrew Wang, Yi Lu
"Kinetic and crystallographic studies of a redesigned manganese-binding site in cytochrome c peroxidase"
Journal of Biological Inorganic Chemistry 12, 126-137 (2007) [doi]

Abstract&nbsp;&nbsp;Manganese peroxidase (MnP) from the white rot fungus Phanerochaete chrysosporium contains a manganese-binding site that plays a critical role in its function. Previously, a MnII-binding site was designed into cytochrome c peroxidase (CcP) based on sequence homology (Yeung et al. in Chem. Biol. 4:215--222, 1997; Gengenbach et al. in Biochemistry 38:11425--11432, 1999). Here, we report a redesign of this site based on X-ray structural comparison of MnP and CcP. The variant, CcP(D37E, V45E, H181E), displays 2.5-fold higher catalytic efficiency (k cat/K M) than the variant in the original design, mostly due to a stronger K M of 1.9&nbsp;mM (vs. 4.1&nbsp;mM). High-resolution X-ray crystal structures of a metal-free form and a form with CoII at the designed MnII site were also obtained. The metal ion in the engineered metal-binding site overlays well with MnII bound in MnP, suggesting that this variant is the closest structural model of the MnII-binding site in MnP for which a crystal structure exists. A major difference arises in the distances of the ligands to the metal; the metal--ligand interactions in the CcP variant are much weaker than the corresponding interactions in MnP, probably owing to partial occupancy of metal ion at the designed site, difference in the identity of metal ions (CoII rather than MnII) and other interactions in the second coordination sphere. These results indicate that the metal ion, the ligands, and the environment around the metal-binding site play important roles in tuning the structure and function of metalloenzymes.
97 | Yi Lu, Juewen Liu
"Functional DNA nanotechnology: emerging applications of DNAzymes and aptamers"
Current Opinion in Biotechnology 17, 580-588 (2006) [doi]

In the past 25 years, DNA molecules have been utilized both as powerful synthetic building blocks to create nanoscale architectures and as versatile programmable templates for assembly of nanomaterials. In parallel, the functions of DNA molecules have been expanded from pure genetic information storage to catalytic functions like those of protein enzymes (DNAzymes) and specific binding functions like antibodies (aptamers). In the past few years, a new interdisciplinary field has emerged that aims to combine functional DNA biology with nanotechnology to generate more dynamic and controllable DNA-based nanostructures or DNA-templated nanomaterials that are responsive to chemical stimuli.
96 | Dewain K. Garner, Mark D. Vaughan, Hee Jung Hwang, Masha G. Savelieff, Steven M. Berry, John F. Honek, Yi Lu
"Reduction Potential Tuning of the Blue Copper Center in Pseudomonas aeruginosa Azurin by the Axial Methionine as Probed by Unnatural Amino Acids"
Journal of the American Chemical Society 128, 15608-15617 (2006) [PDF] [doi]

The conserved axial ligand methionine 121 from Pseudomonas aeruginosa azurin (Az) has been replaced by isostructural unnatural amino acid analogues, oxomethionine (OxM), difluoromethionine (DFM), trifluoromethionine (TFM), selenomethionine (SeM), and norleucine (Nle) using expressed protein ligation. The replacements resulted in <6 nm shifts in the S(Cys)−Cu charge transfer (CT) band in the electronic absorption spectra and <8 gauss changes in the copper hyperfine coupling constants (A∥) in the X-band electron paramagnetic resonance spectra, suggesting that isostructural replacement of Met resulted in minimal structural perturbation of the copper center. The slight blue shifts of the CT band follow the trend of stronger electronegativity of the ligands. This trend is supported by 19F NMR studies of the fluorinated methionine analogues. However, the order of A∥ differs, suggesting additional factors influencing A∥. In contrast to the small changes in the UV−vis and EPR spectra, a large variation of >227 mV in reduction potential was observed for the series of variants reported here. Additionally, a linear correlation was established between the reduction potentials and hydrophobicity of the variants. Extension of this analysis to other type 1 copper-containing proteins reveals a linear correlation between change in hydrophobicity and change in reduction potential, independent of the protein scaffold, experimental conditions, measurement techniques, and steric modifications. This analysis has also revealed for the first time high and low potential states for type 1 centers, and the difference may be attributable to destabilization of the protein fold by disruption of hydrophobic or hydrogen bonding interactions that stabilize the type 1 center.
95 | Juewen Liu, Debapriya Mazumdar, Yi Lu
"A Simple and Sensitive 'Dipstick' Test in Serum Based on Lateral Flow Separation of Aptamer-Linked Nanostructures"
Angewandte Chemie International Edition 45, 7955-7959 (2006) [archived PDF] [doi] [news]

A quick plunge: By using lateral flow devices as a platform to separate aptamer-assembled nanoparticle (NP) aggregates, highly sensitive and selective colorimetric sensors were constructed that mimic litmus paper tests. Detection of cocaine in serum was also realized.
94 | Yi Lu
"Metalloprotein and Metallo-DNA/RNAzyme Design:  Current Approaches, Success Measures, and Future Challenges"
Inorganic Chemistry 45, 9930-9940 (2006) [PDF] [doi]

Specific metal-binding sites have been found in not only proteins but also DNA and RNA molecules. Together these metalloenzymes consist of a major portion of the enzyme family and can catalyze some of the most difficult biological reactions. Designing these metalloenzymes can be both challenging and rewarding because it can provide deeper insights into the structure and function of proteins and cheaper and more stable alternatives for biochemical and biotechnological applications. Toward this goal, both rational and combinatorial approaches have been used. The rational approach is good for designing metalloenzymes that are well characterized, such as heme proteins, while the combinatorial approach is better at designing those whose structures are poorly understood, such as metallo-DNA/RNAzymes. Among the rational approaches, de novo design is at its best when metal-binding sites reside in a scaffold whose structure has been designed de novo (e.g., α-helical bundles). Otherwise, design using native scaffolds can be equally effective, allowing more choices of scaffolds whose structural stability is often more resistant to multiple mutations. In addition, computational and empirical designs have both enjoyed successes. Because of the limitation in defining structural parameters for metal-binding sites, a computational approach is restricted to mostly metal-binding sites that are well defined, such as mono- or homonuclear centers. An empirical approach, even though it is less restrictive in the metal-binding sites to be designed, depends heavily on one's knowledge and choice of templates and targets. An emerging approach is a combination of both computational and empirical approaches. The success of these approaches can be measured not only by three-dimensional structural comparison between the designed and target enzymes but also by the total amount of insight obtained from the design process and studies of the designed enzymes. One of the biggest advantages of designed metalloenzymes is the potential of placing two different metal-binding sites in the same protein framework for comparison. A final measure of success is how one can utilize the insight gained from the intellectual exercise to design new metalloenzymes, including those with unprecedented structures and functions. Future challenges include designing more complex metalloenzymes such as heteronuclear metal centers with strong nanomolar or better affinities. A key to meeting this challenge is to focus on the design of not only primary but also secondary coordination spheres using a combination of improved computer programs, experimental design, and high-resolution crystallography.
93 | Yi Lu
"Metalloprotein Design & Engineering"
in "Encyclopedia of Inorganic Chemistry", Vol. V, R. Bruce King, ed.; John Wiley & Sons, Ltd: Chichester, UK; pp. 3159-3192 (2006) [archived PDF] [doi]

This review covers recent advances in metalloprotein design, with focus on different approaches to the design. Impressive progress has been made in designing metal-binding sites in peptides, de novo designed proteins, and native protein scaffolds. The approach can be rational or combinatorial. Under rational design, redesigning an existing metal-binding site to a new site with dramatically different structure and function complements well the design of new metal-binding sites by revealing the role of specific residues responsible for a particular structural or functional feature of the metal-binding site of interest. To create a new metal-binding site, several approaches have been used, including design based on structural homology, by inspection, using automated computer search algorithms, or combination of the above approaches. In addition, modular approach by transplanting a conserved structural unit from one protein into another has also been shown to be effective. Design through combinatorial and evolution methods has also been successful as it requires little prior knowledge of the protein structure. Finally, introducing unnatural amino acids or nonnative metal ions/prosthetic groups to expand the repertoires of metalloproteins have been demonstrated. Successful examples of each of the approaches are given; advantages and disadvantages of the approaches are discussed; the outlook for future research is also presented.
91 | Yi Lu
"Biosynthetic Inorganic Chemistry"
Angewandte Chemie International Edition 45, 5588-5601 (2006) [doi]

Inorganic chemistry and biology can benefit greatly from each other. Although synthetic and physical inorganic chemistry have been greatly successful in clarifying the role of metal ions in biological systems, the time may now be right to utilize biological systems to advance coordination chemistry. One such example is the use of small, stable, easy-to-make, and well-characterized proteins as ligands to synthesize novel inorganic compounds. This biosynthetic inorganic chemistry is possible thanks to a number of developments in biology. This review summarizes the progress in the synthesis of close models of complex metalloproteins, followed by a description of recent advances in using the approach for making novel compounds that are unprecedented in either inorganic chemistry or biology. The focus is mainly on synthetic ldquotricksrdquo learned from biology, as well as novel structures and insights obtained. The advantages and disadvantages of this biosynthetic approach are discussed.
90 | Juewen Liu, Yi Lu
"Design of asymmetric DNAzymes for dynamic control of nanoparticle aggregation states in response to chemical stimuli"
Organic & Biomolecular Chemistry 4, 3435-3441 (2006) [doi]

Dynamic control of nanomaterial assembly states in response to chemical stimuli is critical in making multi-component materials with interesting properties. Previous work has shown that a Pb2+-specific DNAzyme allowed dynamic control of gold nanoparticle aggregation states in response to Pb2+, and the resulting color change from blue aggregates to red dispersed particles can be used as a convenient way of sensing Pb2+. However, a small piece of DNA (called invasive DNA) and low ionic strength ([similar]30 mM) were required for the process, limiting the scope of application in assembly and sensing. To overcome this limitation, a series of asymmetric DNAzymes, in which one of the two substrate binding regions is longer than the other, has been developed. With such a system, we demonstrated Pb2+-induced disassembly of gold nanoparticle aggregates and corresponding color change at room temperature without the need for invasive DNA, while also making the system more tolerant to ionic strength (33-100 mM). The optimal lengths of the long and short arms were determined to be 14 and 5 base pairs, respectively. In nanoparticle aggregates, the activity of the DNAzyme increased with decreasing ionic strength of the reaction buffer. This simpler and more versatile system allows even better dynamic control of nanoparticle aggregation states in response to chemical stimuli such as Pb2+, and can be used in a wider range of applications for colorimetric sensing of metal ions.
89 | J. Liu, Y. Lu
"Smart Nanomaterials Responsive to Multiple Chemical Stimuli with Controllable Cooperativity"
Advanced Materials 18, 1667-1671 (2006) [doi] [news]

Aptamer-linked smart materials responsive to a combination of any two analytes, including adenosine, cocaine, and K+, are demonstrated. The effect of small molecules on the properties of the nanomaterials can be either highly cooperative or non-cooperative, which can be described as logic gates with chemical inputs and colorimetric output (see figure).
88 | Xuan Zhao, Natasha Yeung, Brandy S. Russell, Dewain K. Garner, Yi Lu
"Catalytic Reduction of NO to N2O by a Designed Heme Copper Center in Myoglobin:  Implications for the Role of Metal Ions"
Journal of the American Chemical Society 128, 6766-6767 (2006) [PDF] [doi]

The effects of metal ions on the reduction of nitric oxide (NO) with a designed heme copper center in myoglobin (F43H/L29H sperm whale Mb, CuBMb) were investigated under reducing anaerobic conditions using UV−vis and EPR spectroscopic techniques as well as GC/MS. In the presence of Cu(I), catalytic reduction of NO to N2O by CuBMb was observed with turnover number of 2 mol NO·mol CuBMb-1·min-1, close to 3 mol NO·mol enzyme-1·min-1 reported for the ba3 oxidases from T. thermophilus. Formation of a His-heme-NO species was detected by UV−vis and EPR spectroscopy. In comparison to the EPR spectra of ferrous-CuBMb-NO in the absence of metal ions, the EPR spectra of ferrous-CuBMb-NO in the presence of Cu(I) showed less-resolved hyperfine splitting from the proximal histidine, probably due to weakening of the proximal His-heme bond. In the presence of Zn(II), formation of a five-coordinate ferrous-CuBMb-NO species, resulting from cleavage of the proximal heme Fe-His bond, was shown by UV−vis and EPR spectroscopic studies. The reduction of NO to N2O was not observed in the presence of Zn(II). Control experiments using wild-type myoglobin indicated no reduction of NO in the presence of either Cu(I) or Zn(II). These results suggest that both the identity and the oxidation state of the metal ion in the CuB center are important for NO reduction. A redox-active metal ion is required to deliver electrons, and a higher oxidation state is preferred to weaken the heme iron−proximal histidine toward a five-coordinate key intermediate in NO reduction.
87 | Juewen Liu, Yi Lu
"Fast Colorimetric Sensing of Adenosine and Cocaine Based on a General Sensor Design Involving Aptamers and Nanoparticles"
Angewandte Chemie International Edition 45, 90-94 (2006) [doi] [news]

Apt to change color: DNA aptamers have been used to assemble DNA-functionalized gold nanoparticles to produce highly sensitive and selective colorimetric sensors with an instantaneous color response on addition of a substrate. The general method has been shown for adenosine and cocaine (see picture), but should be applicable to any aptamer of choice.
86 | Lu Yi
"Catalytic Nucleic Acids (Ribozymes and Deoxyribozymes)"
in "Biological Inorganic Chemistry: Structure & Reactivity", Ivano Bertini, Harry B. Gray, Joan S. Valentine, eds.; University Science Books, Inc.: Mill Valley, CA.; pp. 215-228 (2006) [archived PDF]
85 | Juewen Liu, Yi Lu
"Fluorescent DNAzyme Biosensors for Metal Ions Based on Catalytic Molecular Beacons"
Methods in Molecular Biology 335, (Methods in Molecular Biology) 275-288 (2006)
84 | Juewen Liu, Yi Lu
"Multi-Fluorophore Fluorescence Resonance Energy Transfer for Probing Nucleic Acids Structure and Folding"
Methods in Molecular Biology 335, (Methods in Molecular Biology) 257-271 (2006)
83 | Hee Jung Hwang, Nandini Nagraj, Yi Lu
"Spectroscopic Characterizations of Bridging Cysteine Ligand Variants of an Engineered Cu2(SCys)2 CuA Azurin"
Inorganic Chemistry 45, 102-107 (2006) [PDF] [doi]

Bridging cysteine ligands of the CuA center in an engineered CuA azurin were replaced with serine, and the variants (Cys116Ser and Cys112Ser CuA azurin) were characterized by mass spectrometry, as well as UV-vis and electron paramagnetic resonance (EPR) spectroscopic techniques. The replacements resulted in dramatically perturbed spectroscopic properties, indicating that the cysteines play a critical role in maintaining the structural integrity of the Cu center. The replacements at different cysteine residues resulted in different perturbations, even though the two cysteines are geometrically symmetrical in the primary coordination sphere with respect to the two copper ions. The Cys112Ser variant contains two distinct type 2 copper centers, while the Cys116Ser variant has one type 1 copper center with slight tetragonal distortion. Both the UV-vis and EPR spectra of the Cys116Ser variant change with pH, and the pKa of the transition is 6.0. A type 1 copper EPR spectrum with A? = 26 G was obtained at pH 7.0, while a type 2 copper EPR spectrum with A? = 140 G was found at pH 5.0. Interestingly, lowering the temperature from 290 to 85 K resulted in conversion of the Cys116Ser variant from a type 1 copper center to a type 2 copper center, suggesting rearrangement of the ligand around the copper or binding of an exogenous ligand at low temperature. This difference in mutation effects at different cysteines may be due to different constraints exerted on the two cysteines by hydrogen-bonding patterns in the ligand loop.
82 | Daryl P. Wernette, Carla B. Swearingen, Donald M. Cropek, Yi Lu, Jonathan V. Sweedler, Paul W. Bohn
"Incorporation of a DNAzyme into Au-coated nanocapillary array membranes with an internal standard for Pb(ii) sensing"
The Analyst 131, 41-47 (2006) [doi]

A Pb(ii)-specific DNAzyme has been successfully incorporated into Au-coated polycarbonate track-etched (PCTE) nanocapillary array membranes (NCAMs) by thiol-gold immobilization. Incorporation of the DNAzyme into the membrane provides a substrate-bound sensor using a novel internal control methodology for fluorescence-based detection of Pb(ii). A non-cleavable substrate strand, identical to the cleavable DNAzyme substrate strand except the RNA-base is replaced by the corresponding DNA-base, is used for ratiometric comparison of intensities. The cleavable substrate strand is labeled with fluorescein, and the non-cleavable strand is labeled with a red fluorophore (Cy5 or Alexa 546) for detection after release from the membrane surface. This internal standard based ratiometric method allows for real-time monitoring of Pb(ii)-induced cleavage, as well as standardizing variations in substrate size, solution detection volume, and monolayer density. The result is a Pb(ii)-sensing structure that can be stored in a prepared state for 30 days, regenerated after reaction, and detect Pb(ii) concentrations as low as 17 nM (3.5 ppb).
81 | Ningyan Wang, Xuan Zhao, Yi Lu
"Role of Heme Types in Heme-Copper Oxidases: Effects of Replacing a Heme b with a Heme o Mimic in an Engineered Heme-Copper Center in Myoglobin"
Journal of the American Chemical Society 127, 16541-16547 (2005) [PDF] [doi]

To address the role of the secondary hydroxyl group of heme a/o in heme-copper oxidases, we incorporated Fe(III)-2,4 (4,2) hydroxyethyl vinyl deuterioporphyrin IX, as a heme o mimic, into the engineered heme-copper center in myoglobin (sperm whale myoglobin L29H/F43H, called CuBMb). The only difference between the heme b of myoglobin and the heme o mimic is the substitution of one of the vinyl side chains of the former with a hydroxyethyl group of the latter. This substitution resulted in an  4 nm blue shift in the Soret band and  20 mV decrease in the heme reduction potential. In a control experiment, the heme b in CuBMb was also replaced with a mesoheme, which resulted in an  13 nm blue shift and  30 mV decrease in the heme reduction potential. Kinetic studies of the heme o mimic-substituted CuBMb showed significantly different reactivity toward copper-dependent oxygen reduction from that of the b-type CuBMb. In reaction with O2, CuBMb with a native heme b showed heme oxygenase activity by generating verdoheme in the presence of Cu(I). This heme degradation reaction was slowed by  19-fold in the heme o mimic-substituted CuBMb (from 0.028 s-1 to 0.0015 s-1), while the mesoheme-substituted CuBMb shared a similar heme degradation rate with that of CuBMb (0.023 s-1). No correlation was found between the heme reduction potential and its O2 reactivity. These results strongly suggest the critical role of the hydroxyl group of heme o in modulating heme-copper oxidase activity through participation in an extra hydrogen-bonding network.
80 | Hee Jung Hwang, James R. Carey, Evan T. Brower, Alan J. Gengenbach, Joseph A. Abramite, Yi Lu
"Blue Ferrocenium Azurin: An Organometalloprotein with Tunable Redox Properties"
Journal of the American Chemical Society 127, 15356-15357 (2005) [PDF] [doi]

A ferrocene derivative (2-[(methylsulfonyl)thio]ethylferrocene) (1) has been synthesized and incorporated into apo-azurin from Pseudomonas aeruginosa by covalent attachment to the highly conserved Cys112. The resulting artificial organometalloprotein (a protein containing organometallic compounds in the active site) has been characterized by UV-vis, electrospray mass spectrometry, and cyclic voltammetry (CV). Incorporation of 1 into azurin resulted in a higher solubility of the ferrocene group and improved stability of the ferrocenium species in aqueous solution, as shown by a more intense UV-vis absorption and a more reversible CV of the attached ferrocene group, respectively. The incorporation of 1 also increased the reduction potential of the complex from 402 to 579 mV (vs NHE), consistent with the ferrocene group being encapsulated inside the hydrophobic environment of the protein. Modulation of the reduction potential of ferrocene by residues near the secondary coordination sphere has also been demonstrated. Raising the pH from 4 to 9 resulted in a greater than 80 mV decrease in reduction potential of the protein-bound ferrocene (from 579 to 495 mV), while replacing Met121, an amino acid residue in close proximity to the ferrocene group with a positively charged Arg or negatively charged Glu, resulted in the predicted increase or decrease in reduction potential at all pH values. Similarly, substitution of Met121 with a more hydrophobic Leu raised the reduction potential. The increased solubility, stability, and tune-ability of this organometalloprotein make it an ideal choice for carrying out a number of biological reactions, such as long-range electron transfer or sensing. As an example of such applications, stoichiometric oxidation of ferrocytochrome c by the blue ferrocenium azurin was demonstrated.
79 | Juewen Liu, Yi Lu
"Stimuli-Responsive Disassembly of Nanoparticle Aggregates for Light-Up Colorimetric Sensing"
Journal of the American Chemical Society 127, 12677-12683 (2005) [PDF] [doi]

Controlled assembly of nanomaterials has been the focus of much research. In contrast, controlled disassembly has not received much attention, even though both processes have been shown to be important in biology. By using a Pb2+-dependent RNA-cleaving DNAzyme, we demonstrate here control of the disassembly of gold nanoparticle aggregates in response to Pb2+. In the process, we show that nanoparticle alignment plays an important role in the disassembly process, with the tail-to-tail configuration being the most optimal, probably because of the large steric hindrance of other configurations. The rate of disassembly is significantly accelerated by using small pieces of DNA to invade the cleaved substrate of the DNAzyme. Investigation of such a controlled disassembly process allows the transformation of previously designed light-down colorimetric Pb2+ sensors into light-up sensors.
78 | Tae-Jin Yim, Juewen Liu, Yi Lu, Ravi S. Kane, Jonathan S. Dordick
"Highly Active and Stable DNAzyme-Carbon Nanotube Hybrids"
Journal of the American Chemical Society 127, 12200-12201 (2005) [PDF] [doi]

We report herein the generation and characterization of DNAzyme-carbon nanotube conjugates. Biotinylated DNAzyme was attached to streptavidin-functionalized MWNTs. The resultant DNAzyme-MWNT conjugates exhibited Michaelis-Menten kinetics and multiple catalytic turnovers. The specific activity of the nanoscale conjugates was  1/3 of that for freely soluble DNAzyme. Such high activity may allow myriad applications ranging from the directed assembly of nanomaterials to nanoscale cellular therapeutics that rely on the RNA cleaving ability of DNAzymes.
77 | Juewen Liu, Daryl P. Wernette, Yi Lu
"Proofreading and Error Removal in a Nanomaterial Assembly"
Angewandte Chemie International Edition 44, 7290-7293 (2005) [PDF] [doi]

We all make mistakes! The specific removal of error particles in nanomaterial assemblies is achieved by using a DNAzyme as a proofreading and error-removal unit (see picture of a DNA-templated gold nanoparticle assembly). Parallels can be drawn with the proofreading and error-removal process in biology. A and B=gold nanoparticles attached to DNA sequences; Bprime=error particle.
76 | Haiying Wei, Xiaoyong Wang, Qin Liu, Yuhua Mei, Yi Lu, Zijian Guo
"Disulfide Bond Cleavage Induced by a Platinum(II) Methionine Complex"
Inorganic Chemistry 44, 6077-6081 (2005) [PDF] [doi]

The cleavage of a disulfide bond and the redox equilibrium of thiol/disulfide are strongly related to the levels of glutathione (GSH)/oxidized glutathione (GSSG) or mixed disulfides in vivo. In this work, the cleavage of a disulfide bond in GSSG induced by a platinum(II) complex [Pt(Met)Cl2] (where Met = methionine) was studied and the cleavage fragments or their platinated adducts were identified by means of electrospray mass spectrometry, high-performance liquid chromatography, and ultraviolet techniques. The second-order rate constant for the reaction between [Pt(Met)Cl2] and GSSG was determined to be 0.4 M-1 s-1 at 310 K and pH 7.4, which is 100- and 12-fold faster than those of cisplatin and its monoaqua species, respectively. Different complexes were formed in the reaction of [Pt(Met)Cl2] with GSSG, mainly mono- and dinuclear platinum complexes with the cleavage fragments of GSSG. This study demonstrated that [Pt(Met)Cl2] can promote the cleavage of disulfide bonds. The mechanistic insight obtained from this study may provide a deeper understanding on the potential involvement of platinum complexes in the intracellular GSH/GSSG systems.
75 | In-Hyoung Chang, Joseph J. Tulock, Juewen Liu, Won-Suk Kim, Donald M. Cannon, Yi Lu, Paul W. Bohn, Jonathan V. Sweedler, Donald M. Cropek
"Miniaturized Lead Sensor Based on Lead-Specific DNAzyme in a Nanocapillary Interconnected Microfluidic Device"
Environmental Science & Technology 39, 3756-3761 (2005) [PDF] [doi]

A miniaturized lead sensor has been developed by combining a lead-specific DNAzyme with a microfabricated device containing a network of microfluidic channels that are fluidically coupled via a nanocapillary array interconnect. A DNAzyme construct, selective for cleavage in the presence of Pb2+ and derivatized with fluorophore (quencher) at the 5 (3) end of the substrate and enzyme strands, respectively, forms a molecular beacon that is used as the recognition element. The nanocapillary array membrane interconnect is used to manipulate fluid flows and deliver the small-volume sample to the beacon in a spatially confined detection window where the DNAzyme is interrogated using laser-induced fluorescence detection. A transformed log plot of the fluorescent signal exhibits a linear response (r2 = 0.982) over a Pb2+ concentration range of 0.1-100 M, and a detection limit of 11 nM. The sensor has been applied to the determination of Pb2+ in an electroplating sludge reference material, the result agreeing with the certified value within 4.9%. Quantitative measurement of Pb2+ in this complex sample demonstrates the selectivity of this sensor scheme and points favorably to the application of such technologies to analysis of environmental samples. The unique combination of a DNAzyme with a microfluidic-nanofluidic hybrid device makes it possible to change the DNAzyme to select for other compounds of interest, and to incorporate multiple sensing systems within a single device for greater flexibility. This work represents the initial steps toward creation of a robust field sensor for lead in groundwater or drinking water.
74 | Kevin E. Nelson, Peter J. Bruesehoff, Yi Lu
"In Vitro Selection of High Temperature Zn2+-Dependent DNAzymes"
Journal of Molecular Evolution 61, 216-225 (2005) [PDF] [doi]

In vitro selection of Zn2+-dependent RNA-cleaving DNAzymes with activity at 90\textdegreeC has yielded a diverse spool of selected sequences. The RNA cleavage efficiency was found in all cases to be specific for Zn2+ over Pb2+, Ca2+, Cd2+, Co2+, Hg2+, and Mg2+. The Zn2+-dependent activity assay of the most active sequence showed that the DNAzyme possesses an apparent Zn2+-binding dissociation constant of 234 $\mu$M and that its activity increases with increasing temperatures from 50--90\textdegreeC. A fit of the Arrhenius plot data gave Ea\thinspace=\thinspace15.3 kcal mol?1. Surprisingly, the selected Zn2+-dependent DNAzymes showed only a modest (?3-fold) activity enhancement over the background rate of cleavage of random sequences containing a single embedded ribonucleotide within an otherwise DNA oligonucleotide. The result is attributable to the ability of DNA to sustain cleavage activity at high temperature with minimal secondary structure when Zn2+ is present. Since this effect is highly specific for Zn2+, this metal ion may play a special role in molecular evolution of nucleic acids at high temperature.
73 | Hee Jung Hwang, Steven M. Berry, Mark J. Nilges, Yi Lu
"Axial Methionine Has Much Less Influence on Reduction Potentials in a CuA Center than in a Blue Copper Center"
Journal of the American Chemical Society 127, 7274-7275 (2005) [PDF] [doi]

The role of the highly conserved axial methionine of the purple CuA center in an engineered CuA azurin on modulating the reduction potentials of the copper center was investigated by a systematic replacement of the methionine with glutamate, aspartate, and leucine. In contrast to the same substitutions in the structurally related blue copper azurin, much smaller changes in reduction potential were observed in the CuA azurin upon replacing the methionine ligand with negatively charged Glu (-8 mV) and Asp (-5 mV) and more hydrophobic Leu (+16 mV). These findings are important in understanding the different roles of the two cupredoxins. The diamond core Cu2S2(Cys) structure of the CuA is much more resistant to variations of axial ligand interactions than the distorted tetrahedral structure of the blue copper protein. This difference may translate into a much wider range of reduction potentials (>1000 mV) for blue copper proteins that transfer electrons to a variety of partners in many different biological systems and a much narrower range of reduction potentials (<40 mV) for CuA proteins where a small difference in reduction potentials between the CuA and its redox partners is required.
72 | Lu Yi, Steven M. Berry
"Protein Structure Design and Engineering"
in "Encyclopedia of Life Sciences", John Wiley & Sons, Ltd.: Chichester, UK; (2005) [PDF] [doi]
71 | Xuan Zhao, Mark J. Nilges, Yi Lu
"Redox-Dependent Structural Changes in an Engineered Heme-Copper Center in Myoglobin: Insights into Chloride Binding to CuB in Heme Copper Oxidases"
Biochemistry 44, 6559-6564 (2005) [PDF] [doi]

The effects of chloride on the redox properties of an engineered binuclear heme-copper center in myoglobin (CuBMb) were studied by UV-vis spectroelectrochemistry and EPR spectroscopy. A low-spin heme FeIII-CuI intermediate was observed during the redox titration of CuBMb only in the presence of both CuII and chloride. Upon the first electron transfer to the CuB center, one of the His ligands of CuB center dissociates and coordinates to the heme iron, forming a six-coordinate low-spin ferric heme center and a reduced CuB center. The second electron transfer reduces the ferric heme and causes the release of the coordinated His ligand. Thus, the fully reduced state of the heme-copper center contains a five-coordinate ferrous heme and a reduced CuB center, ready for O2 binding and reduction to water to occur. In the absence of a chloride ion, formation of the low-spin heme species was not observed. These redox reactions are completely reversible. These results indicate that binding of chloride to the CuB center can induce redox-dependent structural changes, and the bound chloride and hydroxide in the heme-copper center may play different roles in the redox-linked enzymatic reactions of heme-copper oxidases, probably because of their different binding affinity to the copper center and the relatively high concentration of chloride under physiological conditions.
70 | Kashan A. Shaikh, Kee Suk Ryu, Edgar D. Goluch, Jwa-Min Nam, Juewen Liu, C. Shad Thaxton, Thomas N. Chiesl, Annelise E. Barron, Yi Lu, Chad A. Mirkin, Chang Liu
"A modular microfluidic architecture for integrated biochemical analysis"
Proceedings of the National Academy of Sciences of the United States of America 102, 9745-9750 (2005) [PDF] [doi]

Microfluidic laboratory-on-a-chip (LOC) systems based on a modular architecture are presented. The architecture is conceptualized on two levels: a single-chip level and a multiple-chip module (MCM) system level. At the individual chip level, a multilayer approach segregates components belonging to two fundamental categories: passive fluidic components (channels and reaction chambers) and active electromechanical control structures (sensors and actuators). This distinction is explicitly made to simplify the development process and minimize cost. Components belonging to these two categories are built separately on different physical layers and can communicate fluidically via cross-layer interconnects. The chip that hosts the electromechanical control structures is called the microfluidic breadboard (FBB). A single LOC module is constructed by attaching a chip comprised of a custom arrangement of fluid routing channels and reactors (passive chip) to the FBB. Many different LOC functions can be achieved by using different passive chips on an FBB with a standard resource configuration. Multiple modules can be interconnected to form a larger LOC system (MCM level). We demonstrated the utility of this architecture by developing systems for two separate biochemical applications: one for detection of protein markers of cancer and another for detection of metal ions. In the first case, free prostate-specific antigen was detected at 500 aM concentration by using a nanoparticle-based bio-bar-code protocol on a parallel MCM system. In the second case, we used a DNAzyme-based biosensor to identify the presence of Pb2+ (lead) at a sensitivity of 500 nM in <1 nl of solution.
69 | Jingwen Chen, Xiaoyong Wang, Yangguang Zhu, Jun Lin, Xiaoliang Yang, Yizhi Li, Yi Lu, Zijian Guo
"An Asymmetric Dizinc Phosphodiesterase Model with Phenolate and Carboxylate Bridges"
Inorganic Chemistry 44, 3422-3430 (2005) [PDF] [doi]

A phosphodiesterase model with two zinc centers has been synthesized and characterized. The compound, [Zn2(L-2H)(AcO)(H2O)](PF6)2H2O (Zn2L), was formed using an end-off type compartmental ligand, 2,6-bis[(2-pyridylmethyl)(2-hydroxyethyl)amino]methyl-4-methylphenol (L), and zinc acetate dihydrate. The X-ray crystallographic analysis shows that Zn2L contains a -acetato--cresolato-dizinc(II) core comprised of a quasi-trigonal bipyramidal Zn and a distorted octahedral Zn, and the distance between them is 3.421 which is close to the dizinc distance in related natural metalloenzymes. Phosphodiesterase activity of Zn2L was investigated using bis(4-nitrophenyl) phosphate (BNPP) as the substrate. The pH dependence of the BNPP cleavage in aqueous buffer media shows a sigmoid-shaped pH-kobs profile with an inflection point around pH 7.13 which is close to the first pKa value of 7.20 for Zn2L obtained from the potentiometric titration. The catalytic rate constant (kcat) is 4.60 10-6 s-1 at pH 7.20 and 50 C which is ca. 105-fold higher than that of the uncatalyzed reaction. The deprotonated alcoholic group appended on Zn2L is responsible for the cleavage reaction. The possible mechanism for the BNPP cleavage promoted by Zn2L is proposed on the basis of kinetic and spectral analysis. The dizinc complex formed in situ in anhydrous DMSO exhibits a similar ability to cleave BNPP. This study provides a less common example for the phosphodiesterase model in which the metal-bound alkoxide is the nucleophile.
68 | Jennifer L. Seifert, Thomas D. Pfister, Judith M. Nocek, Yi Lu, Brian M. Hoffman
"Hopping in the Electron-Transfer Photocycle of the 1:1 Complex of Zn-Cytochrome c Peroxidase with Cytochrome c"
Journal of the American Chemical Society 127, 5750-5751 (2005) [PDF] [doi]

The physiological electron-transfer (ET) partners, cytochrome c peroxidase (CcP) and cytochrome c (Cc)1, can be modified to exhibit photoinitiated ET through substitution of Zn (or Mg) for Fe in either partner. Laser excitation of the Zn-porphyrin (ZnP) to its triplet excited state (3ZnP) initiates direct heme-heme ET to the ferriheme center of its partner across the protein-protein interface. This photoinitiated ET produces the charge-separated intermediate, I = [ZnP+CcP, Fe2+Cc], with a metalloporphyrin p-cation radical (ZnP+) in the donor protein and a ferroheme acceptor protein. I, in general, is thought to return to the ground state by a thermal ET process that involves direct heme-heme back-ET to complete a simple photocycle. We here contrast intracomplex ET between yeast iso-1 Cc and ZnCcP(WT) (wild-type) with that for two ZnCcP(X) variants:? X = W191F, with redox-active W191 replaced by Phe; WYM4, a W191F mutant with further replacement of four other potentially redox-active sites (W51F, Y187F, Y229F, and Y236F). The results show that W191 acts as an ET mediator, which short-circuits the direct heme-heme back-ET through a two-step, hopping process in which the ZnP+ cation radical formed by photoinitiated ET rapidly oxidizes W191, and the resultant W191+, in turn, rapidly oxidizes Fe2+Cc.
67 | Yi Lu
"Design and engineering of metalloproteins containing unnatural amino acids or non-native metal-containing cofactors"
Current Opinion in Chemical Biology 9, 118-126 (2005) [PDF] [doi]

An emerging branch of metalloprotein design and engineering is on the horizon, where unnatural amino acids or non-native metal-containing cofactors are employed in the design and engineering process. These endeavors have been shown to be quite effective in elucidating the precise roles of key residues in protein structures and functions, in providing guiding principles on protein design, in fine-tuning the protein properties to an unprecedented level, and in expanding the repertoire of protein functionalities, and thus its range of applications.
66 | Thomas D. Pfister, Takahiro Ohki, Takafumi Ueno, Isao Hara, Seiji Adachi, Yumiko Makino, Norikazu Ueyama, Yi Lu, Yoshihito Watanabe
"Monooxygenation of an Aromatic Ring by F43W/H64D/V68I Myoglobin Mutant and Hydrogen Peroxide"
Journal of Biological Chemistry 280, 12858-12866 (2005) [PDF] [doi]

Myoglobin (Mb) is used as a model system for other heme proteins and the reactions they catalyze. The latest novel function to be proposed for myoglobin is a P450 type hydroxylation activity of aromatic carbons (Watanabe, Y., and Ueno, T. (2003) Bull. Chem. Soc. Jpn. 76, 13091322). Because Mb does not contain a specific substrate binding site for aromatic compounds near the heme, an engineered tryptophan in the heme pocket was used to model P450 hydroxylation of aromatic compounds. The monooxygenation product was not previously isolated because of rapid subsequent oxidation steps (Hara, I., Ueno, T., Ozaki, S., Itoh, S., Lee, K., Ueyama, N., and Watanabe, Y. (2001) J. Biol. Chem. 276, 3606736070). In this work, a Mb variant (F43W/H64D/V68I) is used to characterize the monooxygenated intermediate. A modified (+16 Da) species forms upon the addition of 1 eq of H2O2. This product was digested with chymotrypsin, and the modified peptide fragments were isolated and characterized as 6-hydroxytryptophan using matrix-assisted laser desorption ionization time-of-flight tandem mass spectroscopy and 1H NMR. This engineered Mb variant represents the first enzyme to preferentially hydroxylate the indole side chain of Trp at the C6 position. Finally, heme extraction was used to demonstrate that both the formation of the 6-hydroxytryptophan intermediate (+16 Da) and subsequent oxidation to form the +30 Da final product are catalyzed by the heme cofactor, most probably via the compound I intermediate. These results provide insight into the mechanism of hydroxylation of aromatic carbons by heme proteins, demonstrating that non-thiolate-ligated heme enzymes can perform this function. This establishes Mb compound I as a model for P450 type aromatic hydroxylation chemistry.
65 | Xuan Zhao, Natasha Yeung, Zhilin Wang, Zijian Guo, Yi Lu
"Effects of Metal Ions in the CuB Center on the Redox Properties of Heme in Heme-Copper Oxidases: Spectroelectrochemical Studies of an Engineered Heme-Copper Center in Myoglobin"
Biochemistry 44, 1210-1214 (2005) [PDF] [doi]

The electrochemical properties of an engineered heme-copper center in myoglobin have been investigated by UV-visible spectroelectrochemistry. In the cyanide-bridged, spin-coupled heme-copper center in an engineered myoglobin, the presence of Zn(II) in the CuB center raises the heme reduction potential from -85 to 49 mV vs NHE. However, in the cyanide-free, spin-decoupled derivative of the same protein, the presence of Zn(II) in the CuB center exerts little influence on the heme reduction potentials (77 and 80 mV vs NHE, respectively, in the absence and in the presence of Zn(II)). Similar trends have also been observed when copper ion is present in the CuB center, although on a smaller scale, due to reduction of Cu(II) to Cu(I) prior to heme reduction. These results show that the presence of a metal ion in the designed CuB center has a significant effect on the redox potential of heme Fe only when the two metal centers are coupled through a bridging ligand between the two metal centers, indicating that spin coupling plays an important role in redox potential regulation. In addition, the presence of a single positively charged Cu(I) center in the CuB center resulted in a much lower increase (16 mV) in heme reduction potential than that of two positively charged Zn(II) (118 mV). Therefore, the heme reduction potential must be lowered after the first electron transfer to reduce heme Fe3+-CuB2+ to Fe3+-CuB+. To raise the heme reduction potential to make the second electron transfer (i.e., reduction of Fe3+-CuB+ to Fe2+-CuB+) to be favorable, most likely a proton or decoupling of the heme-copper center is needed in the heme-copper site. These findings provide a strong argument for a thermodynamic driving force basis for redox-regulated proton transfer in heme-copper oxidases.
64 | Carla B. Swearingen, Daryl P. Wernette, Donald M. Cropek, Yi Lu, Jonathan V. Sweedler, Paul W. Bohn
"Immobilization of a Catalytic DNA Molecular Beacon on Au for Pb(II) Detection"
Analytical Chemistry 77, 442-448 (2005) [PDF] [doi]

A Pb(II)-specific DNAzyme fluorescent sensor has been modified with a thiol moiety in order to immobilize it on a Au surface. Self-assembly of the DNAzyme is accomplished by first adsorbing the single-thiolated enzyme strand (HS-17E-Dy) followed by adsorption of mercaptohexanol, which serves to displace any Au-N interactions and ensure that DNA is bound only through the S-headgroup. The preformed self-assembled monolayer is then hybridized with the complementary fluorophore-containing substrate strand (17DS-Fl). Upon reaction with Pb(II), the substrate strand is cleaved, releasing a fluorescent fragment for detection. Fluorescence intensity may be correlated with original Pb(II) concentration, and a linear calibration was obtained over nearly four decades:? 10 M = [Pb(II)] = 1 nM. The immobilized DNAzyme is a robust system; it may be regenerated after cleavage, allowing multiple sensing cycles. In addition, drying of fully assembled DNAzyme before reaction with Pb(II) does not significantly affect analytical performance. These results demonstrate that, in comparison with solution-based schemes, immobilization of the DNAzyme sensor onto a Au surface lowers the detection limit (from 10 to 1 nM), maintains activity and specificity, and allows sensor regeneration and long-term storage. Realization of Pb(II) detection through an immobilized DNAzyme is the first important step toward creation of a stand-alone, portable Pb(II) detection device such as those immobilizing DNAzyme recognition motifs in the nanofluidic pores of a microfluidic-nanofluidic hybrid multilayer device.
63 | Juewen Liu, Yi Lu
"Accelerated Color Change of Gold Nanoparticles Assembled by DNAzymes for Simple and Fast Colorimetric Pb2+ Detection"
Journal of the American Chemical Society 126, 12298-12305 (2004) [PDF] [doi]

The combination of high metal selectivity of DNAzymes with the strong distance-dependent optical properties of metallic nanoparticles has presented considerable opportunities for designing colorimetric sensors for metal ions. We previously communicated a design for a colorimetric lead sensor based on the assembly of gold nanoparticles by a Pb2+-dependent DNAzyme. However, heating to 50 C followed by a cooling process of  2 h was required to observe the color change. Herein we report a new improved design that allows fast (<10 min) detection of Pb2+ at ambient temperature. This improvement of sensor performance is a result of detailed studies of the DNAzyme and nanoparticles, which identified tail-to-tail nanoparticle alignment, and large (42 nm diameter) nanoparticle size as the major determining factors in allowing fast color changes. The optimal conditions for other factors such as temperature (35 C) and concentrations of the DNAzyme (2 M), its substrate (3 nM), and NaCl (300 mM) have also been determined. These results demonstrate that fundamental understanding of the DNAzyme biochemistry and nanoparticle science can lead to dramatically improved colorimetric sensors.
62 | Hee Jung Hwang, Yi Lu
"pH-dependent transition between delocalized and trapped valence states of a CuA center and its possible role in proton-coupled electron transfer"
Proceedings of the National Academy of Sciences of the United States of America 101, 12842-12847 (2004) [PDF] [doi]

A pH-dependent transition between delocalized and trapped mixed valence states of an engineered CuA center in azurin has been investigated by UV-visible absorption and electron paramagnetic resonance spectroscopic techniques. At pH 7.0, the CuA azurin displays a typical delocalized mixed valence dinuclear [Cu(1.5)Cu(1.5)] spectra with optical absorptions at 485, 530, and 760 nm, and with a seven-line EPR hyperfine. Upon lowering of the pH from 7.0 to 4.0, the absorption at 760 nm shifted to lower energy toward 810 nm, and a four-line EPR hyperfine, typical of a trapped valence, was observed. The pH-dependent transition is reversible because increasing the pH restores all delocalized spectral features. Lowering the pH resulted in not only a trapped valence state, but also a dramatically increased reduction potential of the Cu center (from 160 mV to 340 mV). Mutation of the titratable residues around the metal-binding site ruled out Glu-114 and identified the C-terminal histidine ligand (His-120) as a site of protonation, because the His120Ala mutation abolished the above pH-dependent transition. The corresponding histidine in cytochrome c oxidases is along a major electron transfer pathway from CuA center to heme a. Because the protonation of this histidine can result in an increased reduction potential that will prevent electron flow from the CuA to heme a, the CuA and the histidine may play an important role in regulating proton-coupled electron transfer.
61 | Juewen Liu, Yi Lu
"Optimization of a Pb2+-Directed Gold Nanoparticle/DNAzyme Assembly and Its Application as a Colorimetric Biosensor for Pb2+"
Chemistry of Materials 16, 3231-3238 (2004) [PDF] [doi]

We previously communicated a method for directed assembly of gold nanoparticles using a Pb2+-dependent DNAzyme and demonstrated the application of this system as a colorimetric biosensor. The sensor shows high sensitivity and selectivity toward Pb2+ and undergoes a blue-to-red color transition in the presence of Pb2+. To gain a deeper insight into the analyte-directed nanomaterials assembly and sensing processes, a detailed characterization of the system has been performed. First, we found that the presence of gold nanoparticles had no effect on the Pb2+-dependent activity of the DNAzyme and the presence of DNAzyme has little effect on the melting properties of the DNA-functionalized nanoparticle aggregates, suggesting that the performance of the nanoparticle and DNAzyme systems can be optimized independently. Second, the optimal length of the DNAzyme and the alignment of the DNA-functionalized gold nanoparticles for the assembly and sensing processes have been determined to be 9 base pairs on each end for the DNAzyme, and head-to-tail alignment for the DNA-functionalized gold nanoparticles. Third, the optimal stoichiometry of the enzyme to the substrate strands of the DNAzyme was shown to be one to one in nanoparticle aggregates. Finally, the most favorable temperature and pH conditions for the system have also been established, with a temperature of 37 C and pH of 6.4 to 9.2 as the best operating conditions. The study also revealed that, for most efficient assembly of nanoparticles, the DNA backbone should be rigidified by formation of a double helix with other DNA molecules. These findings allow optimization of the processes for directed assembly of nanomaterials and for colorimetric sensing.
60 | James R. Carey, Steven K. Ma, Thomas D. Pfister, Dewain K. Garner, Hyeon K. Kim, Joseph A. Abramite, Zhilin Wang, Zijian Guo, Yi Lu
"A Site-Selective Dual Anchoring Strategy for Artificial Metalloprotein Design"
Journal of the American Chemical Society 126, 10812-10813 (2004) [PDF] [doi]

Introducing nonnative metal ions or metal-containing prosthetic groups into a protein can dramatically expand the repertoire of its functionalities and thus its range of applications. Particularly challenging is the control of substrate-binding and thus reaction selectivity such as enantioselectivity. To meet this challenge, both non-covalent and single-point attachments of metal complexes have been demonstrated previously. Since the protein template did not evolve to bind artificial metal complexes tightly in a single conformation, efforts to restrict conformational freedom by modifying the metal complexes and/or the protein are required to achieve high enantioselectivity using the above two strategies. Here we report a novel site-selective dual anchoring (two-point covalent attachment) strategy to introduce an achiral manganese salen complex (Mn(salen)), into apo sperm whale myoglobin (Mb) with bioconjugation yield close to 100%. The enantioselective excess increases from 0.3% for non-covalent, to 12.3% for single point, and to 51.3% for dual anchoring attachments. The dual anchoring method has the advantage of restricting the conformational freedom of the metal complex in the protein and can be generally applied to protein incorporation of other metal complexes with minimal structural modification to either the metal complex or the protein.
59 | Juewen Liu, Yi Lu
"Colorimetric Biosensors Based on DNAzyme-Assembled Gold Nanoparticles"
Journal of Fluorescence 14, 343-354 (2004) [PDF] [doi]

Taking advantage of recent developments in the field of metallic nanoparticle-based colorimetric DNA detection and in the field of in vitro selection of functional DNA/RNA that can recognize a wide range of analytes, we have designed highly sensitive and selective colorimetric biosensors for many analytes of choice. As an example of the sensor design strategy, a highly sensitive and selective colorimetric lead biosensor based on DNAzyme-directed assembly of gold nanoparticles is reviewed. The DNAzyme consists of an enzyme and a substrate strand, which can be used to assemble DNA-functionalized gold nanoparticles. The aggregation brings gold nanoparticles together, resulting in a blue-colored nanoparticle assembly. In the presence of lead, the DNAzyme catalyzes specific hydrolytic cleavage of the substrate strand, which disrupts the formation of the nanoparticle assembly, resulting in red-colored individual nanoparticles. The application of the sensor in lead detection in leaded paint is also demonstrated. In perspective, the use of allosteric DNA/RNAzymes to expand the range of the nanoparticle-based sensor design method is described.
58 | HeeJung Hwang, Majorie Ang, Yi Lu
"Determination of reduction potential of an engineered Cu A azurin by cyclic voltammetry and spectrochemical titrations"
Journal of Biological Inorganic Chemistry 9, 489-494 (2004) [PDF] [doi]

The reduction potentials of an engineered Cu A azurin in its native and thermally denatured states have been determined using cyclic voltammetry and spectrochemical titrations. Using a 4,4'-dipyridyl disulfide modified gold electrode, the reduction potentials of native and thermally denatured Cu A azurin are the same within the experimental error (422\textpm5 and 425\textpm5?mV vs. NHE, respectively, in 50?mM ammonium acetate buffer, pH?5.1, 300?mM NaCl, 25?\textdegreeC), indicating that the potential is that of a nonnative state. In contrast, using a didodecyldimethylammonium bromide (DDAB) film-pyrolytic graphite edge (PGE) electrode, the reduction potentials of native and thermally denatured Cu A azurin have been determined to be 271\textpm7?mV (50?mM ammonium acetate buffer, pH?5.1, 4?\textdegreeC) and 420\textpm1?mV (50?mM ammonium acetate buffer, pH?5.1, 25?\textdegreeC), respectively. Spectroscopic redox titration using [Ru(NH 3) 5Py] 2+ resulted in a reduction potential (254\textpm4?mV) (50?mM ammonium acetate buffer, pH?5.1, 4?\textdegreeC) similar to the value obtained using the DDAB film-PGE electrochemical method. Complete reoxidation of [Ru(NH 3) 5Py] 2+-reduced Cu A azurin is also consistent with the conclusion that this spectrochemical titration method using [Ru(NH 3) 5Py] 2+ measures the reduction potential of native Cu A azurin.
57 | Martina Ralle, Steven M. Berry, Mark J. Nilges, Matt D. Gieselman, Wilfred A. van der Donk, Yi Lu, Ninian J. Blackburn
"The Selenocysteine-Substituted Blue Copper Center:  Spectroscopic Investigations of Cys112SeCys Pseudomonas aeruginosa Azurin"
Journal of the American Chemical Society 126, 7244-7256 (2004) [PDF] [doi]

Azurin is a small electron-transfer protein belonging to the cupredoxin family. The Cu atom is located within a trigonal plane coordinated by two histidines (His46 and His117) and a cysteine (Cys112) with two more distant ligands (Gly45 and Met121) providing axial interactions. A Cys112SeCys derivative has been prepared by expressed protein ligation, and detailed UV/vis, EPR and EXAFS studies at the Cu and Se K-edges have been carried out. Marked changes are observed between the EPR parameters of the Cys112SeCys and WT azurin derivatives, which include a 2-fold increase in A||, a decrease in g-values, and a large increase in rhombicity of the g-tensor. The Cu−Se and Se−Cu bond lengths obtained from analysis of the Cu and Se K-EXAFS of the oxidized protein were found to be 2.30 and 2.31 Å, respectively, 0.14 Å longer than the Cu−S distance of the WT protein. Unexpectedly, the Cu−Se bond lengths were found to undergo only minor changes during reduction, suggesting a very similar structure in both redox states and extending the “rack” hypothesis to the Se-substituted protein.
56 | Juewen Liu, Yi Lu
"Adenosine-Dependent Assembly of Aptazyme-Functionalized Gold Nanoparticles and Its Application as a Colorimetric Biosensor"
Analytical Chemistry 76, 1627-1632 (2004) [PDF] [doi]

Previous work has shown that DNAzyme-directed assembly of gold nanoparticles can be utilized to make effective colorimetric biosensors. However, the method is restricted to analytes that are directly involved in phosphodiester cleavage. To expand the methodology to a broader range of analytes, a colorimetric adenosine biosensor based on the aptazyme-directed assembly of gold nanoparticles is reported here. The aptazyme is based on the 8−17 DNAzyme with an adenosine aptamer motif that can modulate the DNAzyme activity through allosteric interactions depending on the presence of adenosine. In the absence of adenosine, the aptazyme is inactive and the substrate strands can serve as linkers to assemble DNA-functionalized 13-nm-diameter gold nanoparticles, resulting in a blue color. However, the presence of adenosine activates the aptazyme, which cleaves the substrate strand, disrupting the formation of nanoparticle aggregates. A red color of separated gold nanoparticles is observed. Concentrations of adenosine of up to 1 mM can be measured semiquantitatively by the degree of blue to red color changes or quantitatively by the extinction ratio at 520 and 700 nm. Under the same conditions, 5 mM guanosine, cytidine, or uridine resulted in a blue color only, indicating good selectivity of the sensor. The color difference can be clearly observed by the naked eye by spotting the resulting sensor solution onto an alumina TLC plate. Since aptamers that can target many classes of important analytes have already been selected, they can be adapted into aptazyme systems through rational design or further selection. Thus, colorimetric biosensors for many analytes of interest can be designed using the method presented here, regardless of whether the analytes are directly involved in the cleavage reaction or not.
55 | Hee Jung Hwang, Yi Lu
"Spectroscopic evidence for interactions between hexacyanoiron(II/III) and an engineered purple CuA azurin"
Journal of Inorganic Biochemistry 98, 797 - 802 (2004) [PDF] [doi]

Interactions between hexacyanoiron(II/III) and a dinuclear, mixed valence CuA center in engineered CuA azurin have been investigated by UV-visible (UV-vis) and electron paramagnetic resonance (EPR) spectroscopic techniques. Addition of ferricyanide (hexacyanoiron(III)) to the CuA azurin resulted in a new absorption band around 500 nm in the UV-vis and an isotropic line at g=2.16 in the EPR spectra. Control experiments, including additions of Cu(II)SO4 or Cu(I)(CH3CN)4PF6 to ferricyanide or ferrocyanide, as well as gel filtration purification of the ferricyanide-CuA azurin adduct indicate complex formation between cupric ion and ferrocyanide ion in the protein. Solvent or small molecule accessibility, metal oxidation state and the presence of more than one metal ion are potential factors important for the complex formation. These findings must be taken into consideration when using ferricyanide or ferrocyanide as redox agents for studying CuA centers in proteins.
54 | Juewen Liu, Yi Lu
"Improving Fluorescent DNAzyme Biosensors by Combining Inter- and Intramolecular Quenchers"
Analytical Chemistry 75, 6666-6672 (2003) [PDF] [doi]

A previously reported DNAzyme-based biosensor for Pb2+ has shown high sensitivity and selectivity at 4 °C. In the system, the substrate and the enzyme strand of the DNAzyme are labeled with a fluorophore and a quencher, respectively. In the presence of Pb2+, the substrate strand is cleaved by the enzyme strand, and the release of the cleaved fragment results in significant fluorescence increase. However, the performance of the sensor decreases considerably if the temperature is raised to room temperature because of high background fluorescence. A careful analysis of the sensor system, including measurement of the melting curve and fluorescence resonance energy-transfer (FRET) study of the free substrate, suggests that a fraction of the fluorophore-labeled substrate strand is dissociated from the enzyme strand, resulting in elevated background fluorescence signals at room temperature. To overcome this problem, we designed a new sensor system by introducing both inter- and intramolecular quenchers. The design was aided by the FRET study that showed the dissociated substrate maintained a random coil conformation with an end-to-end distance of ∼39 Å, which is much shorter than that of the fully extended DNA. With this new design, the background fluorescence was significantly suppressed, with 660% increase of fluorescence intensity as compared to 60% increase for the previous design. This suppression of background fluorescence signals was achieved without losing selectivity of the sensor. The new design makes it possible to use the sensor for practical applications in a wide temperature range. The design principle presented here should be applicable to other nucleic acid-based biosensors to decrease background fluorescence.
53 | Steven M. Berry, Martina Ralle, Donald W. Low, Ninian J. Blackburn, Yi Lu
"Probing the Role of Axial Methionine in the Blue Copper Center of Azurin with Unnatural Amino Acids"
Journal of the American Chemical Society 125, 8760-8768 (2003) [PDF] [doi]

Expressed protein ligation was used to replace the axial methionine of the blue copper protein azurin from Pseudomonas aeruginosa with unnatural amino acids. The highly conserved methionine121 residue was replaced with the isostructural amino acids norleucine (Nle) and selenomethionine (SeM). The UV−visible absorption, X- and Q-band EPR, and Cu EXAFS spectra of the variants are slightly perturbed from WT. All variants have a predominant S(Cys) to Cu(II) charge transfer band around 625 nm and narrow EPR hyperfine splittings. The Se EXAFS of the M121SeM variant is also reported. In contrast to the small spectral changes, the reduction potentials of M121SeM, M121Leu, and M121Nle are 25, 135, and 140 mV, respectively, higher than that of WT azurin. The use of unnatural amino acids allowed deconvolution of different factors affecting the reduction potentials of the blue copper center. A careful analysis of the WT azurin and its variants obtained in this work showed the large reduction potential variation was linearly correlated with the hydrophobicity of the axial ligand side chains. Therefore, hydrophobicity is the dominant factor in tuning the reduction potentials of blue copper centers by axial ligands.
52 | Juewen Liu, Yi Lu
"A Colorimetric Lead Biosensor Using DNAzyme-Directed Assembly of Gold Nanoparticles"
Journal of the American Chemical Society 125, 6642-6643 (2003) [PDF] [doi]

A highly sensitive and selective colorimetric lead biosensor based on DNAzyme-directed assembly of gold nanoparticles is reported. It consists of a DNAzyme and its substrate that can hybridize to a 5‘-thio-modified DNA attached to gold nanoparticles. The hybridization brings gold nanoparticles together, resulting in a blue-colored nanoparticle assembly. In the presence of lead, the DNAzyme catalyzes specific hydrolytic cleavage, which prevents the formation of the nanoparticle assembly, resulting in red-colored individual nanoparticles. The detection level can be tuned to several orders of magnitude, from 100 nM to over 200 μM, through addition of an inactive variant of the DNAzyme. The concept developed here can be applied to the design of nucleic acid enzyme/nanoparticle sensors for analytes that are subject to in vitro selection, and thus can significantly expand the scope of nanomaterial applications and provide a novel approach to designing simple colorimetric biosensors.
51 | Andrea K. Brown, Jing Li, Caroline M.-B. Pavot, Yi Lu
"A Lead-Dependent DNAzyme with a Two-Step Mechanism†"
Biochemistry 42, 7152-7161 (2003) [PDF] [doi]

A detailed biochemical and mechanistic study of in vitro selected variants of 8−17 DNAzymes is presented. Even though the 8−17 DNAzyme motif has been obtained through in vitro selection under three different conditions involving 10 mM Mg2+ (called 8−17), 0.5 mM Mg2+/50 mM histidine (called Mg5), or 100 μM Zn2+ (called 17E), all variants are shown to be the most active with Pb2+ (8−17:  kobs ∼0.5 min-1; Mg5:  kobs ∼2 min-1; 17E:  kobs ∼1 min-1 with 200 μM Pb2+ at pH 5.0). For the 17E variant of the 8−17 DNAzyme, the single-turnover rate constants followed the order of Pb2+ ≫ Zn2+ ≫ Mn2+ ≈ Co2+ > Ni2+ > Mg2+ ≈ Ca2+ > Sr2+ ≈ Ba2+. The catalytic rate is half-maximal at 13.5 μM Pb2+, 0.97 mM Zn2+, or 10.5 mM Mg2+, suggesting that the metal-binding affinity of the DNAzymes is in the order of Pb2+ > Zn2+ > Mg2+. The Pb2+-dependent activity increases linearly with pH and the slope of the plot of log kobs versus pH is ∼1, suggesting a single deprotonation in the rate-limiting step of the reaction. Sequence variations of the DNAzyme confirm the importance of the G•T wobble pair, the two loops and the intervening stem in maintaining the active conformation of the system. While Mg2+ and Zn2+ catalyze only a transesterification reaction with formation of a product containing a 2‘,3‘-cyclic phosphate, Pb2+ catalyzes a transesterification reaction followed by hydrolysis of the 2‘,3‘-cyclic phosphate. Although this two-step mechanism has shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Therefore, the two-step mechanism is observed in metalloenzymes of all classes, and this 8−17 DNAzyme provides a simple, stable, and cost-effective model system for understanding the structure of Pb2+-binding sites and their roles in the two-step mechanism.
50 | Y. Lu
"Electron Transfer: Cupredoxins"
in "Comprehensive Coordination Chemistry II", Vol. 8, J. A. McCleverty, T. J. Meyer, eds.; Pergamon: Oxford; pp. 91-122 (2003) [PDF] [doi]
49 | Manliang Feng, Hiroyasu Tachikawa, Xiaotang Wang, Thomas Pfister, Alan Gengenbach, Yi Lu
"Resonance Raman spectroscopy of cytochrome c peroxidase variants that mimic manganese peroxidase"
Journal of Biological Inorganic Chemistry 8, 699-706 (2003) [PDF] [doi]

Cytochrome c peroxidase (CcP) variants with an engineered Mn(II) binding site, including MnCcP [CcP(MI, G41E, V45E, H181D)], MnCcP(W191F), and MnCcP(W191F, W51F), that mimic manganese peroxidase (MnP), have been characterized by resonance Raman (RR) spectroscopy. Analysis of the Raman bands in the 200--700?cm?1 and 1300--1650?cm?1 regions indicates that both the coordination and spin state of the heme iron in the variants differ from that of CcP(MI), the recombinant yeast CcP containing additional Met-Ile residues at the N-terminus. At neutral pH the frequencies of the $\nu$3 mode indicate that a pure five-coordinate heme iron exists in CcP(MI) whereas a six-coordinate low-spin iron is the dominant species in the CcP variants with the engineered Mn(II) binding site. The H181D mutation, which weakens the proximal linkage to the heme iron, may be responsible for these spectral and structural changes. Raman spectra of the variants CcP(MI, W191F) and CcP(MI, W191F, W51F) were also obtained to clarify the structural and functional roles of mutations at two tryptophan sites. The W51F mutation was found to disrupt H-bonding to the distal water molecules and the resulting variants tended to form transitional or mixed coordination states that possess spectral and structural features similar to that of MnP. Such structural features, with a loosened distal water, may facilitate the binding of H2O2 and increase the rate constant for compound I formation. This effect, in addition to the elimination of an H-bond to ferryl oxygen by the same mutation, accounts for the increased MnP specific activity of MnCcP(W191F, W51F).
48 | Jeffrey A. Sigman, Hyeon K. Kim, Xuan Zhao, James R. Carey, Yi Lu
"The role of copper and protons in heme-copper oxidases: Kinetic study of an engineered heme-copper center in myoglobin"
Proceedings of the National Academy of Sciences of the United States of America 100, 3629-3634 (2003) [PDF] [doi]

To probe the role of copper and protons in heme-copper oxidase (HCO), we have performed kinetic studies on an engineered heme-copper center in sperm whale myoglobin (Leu-29 → His/Phe-43 → His, called CuBMb) that closely mimics the heme-copper center in HCO. In the absence of metal ions, the engineered CuB center in CuBMb decreases the O2 binding affinity of the heme. However, addition of Ag(I), a redox-inactive mimic of Cu(I), increases the O2-binding affinity. More importantly, copper ion in the CuB center is essential for O2 reduction, as no O2 reduction can be observed in copper-free, Zn(II), or Ag(I) derivatives of CuBMb. Instead of producing a ferryl-heme as in HCO, the CuBMb generates verdoheme because the engineered CuBMb may lack a hydrogen bonding network that delivers protons to promote the heterolytic O—O cleavage necessary for the formation of ferryl-heme. Reaction of oxidized CuBMb with H2O2, a species equivalent in oxidation state to 2e−, reduced O2 but, possessing the extra protons, resulted in ferryl-heme formation, as in HCO. The results showed that the CuB center plays a critical role in O2 binding and reduction, and that proton delivery during the O2 reduction is important to avoid heme degradation and to promote the HCO reaction.
47 | Roshan Perera, Masanori Sono, Jeffrey A. Sigman, Thomas D. Pfister, Yi Lu, John H. Dawson
"Neutral thiol as a proximal ligand to ferrous heme iron: Implications for heme proteins that lose cysteine thiolate ligation on reduction"
Proceedings of the National Academy of Sciences of the United States of America 100, 3641-3646 (2003) [PDF] [doi]

Cysteine plays a key role as a metal ligand in metalloproteins. In all well-recognized cases, however, it is the anionic cysteinate that coordinates. Several cysteinate-ligated heme proteins are known, but some fail to retain thiolate ligation in the ferrous state, possibly following protonation to form neutral cysteine. Ligation by cysteine thiol in ferrous heme proteins has not been documented. To establish spectroscopic signatures for such systems, we have prepared five-coordinate adducts of the ferrous myoglobin H94G cavity mutant with neutral thiol and thioether sulfur donors as well as six-coordinate derivatives such as with CO and, when possible, with NO and O2. A thiol-ligated oxyferrous complex is reported, to our knowledge for the first time. Further, a bis-thioether ferrous H93G model for bis-methionine ligation, as found in Pseudomonas aeruginosa bacterioferritin heme protein, is described. Magnetic CD spectroscopy has been used due to its established ability in axial ligand identification. The magnetic CD spectra of the H93G complexes have been compared with those of ferrous H175C/D235L cytochrome c peroxidase to show that its proximal ligand is neutral cysteine. We had previously reported this cytochrome c peroxidase mutant to be cysteinate-ligated in the ferric state, but the ferrous ligand was undetermined. The spectral properties of ferrous liver microsomal cytochrome P420 (inactive P450) are also consistent with thiol ligation. This study establishes that neutral cysteine can serve as a ligand in ferrous heme iron proteins, and that ferric cysteinate-ligated heme proteins that fail to retain such ligation on reduction may simply be ligated by neutral cysteine.
46 | Yi Lu, Juewen Liu, Jing Li, Peter J. Bruesehoff, Caroline M. -B. Pavot, Andrea K. Brown
"New highly sensitive and selective catalytic DNA biosensors for metal ions"
Biosensors and Bioelectronics 18, 529 - 540 (2003) [PDF] [doi]

While remarkable progress has been made in developing sensors for metal ions such as Ca(II) and Zn(II), designing and synthesizing sensitive and selective metal ion sensors remains a significant challenge. Perhaps the biggest challenge is the design and synthesis of a sensor capable of specific and strong metal binding. Since our knowledge about the construction of metal-binding sites in general is limited, searching for sensors in a combinatorial way is of significant value. Therefore, we have been able to use a combinatorial method called in vitro selection to obtain catalytic DNA that can bind a metal ion of choice strongly and specifically. The metal ion selectivity of the catalytic DNA was further improved using a [`]negative selection' strategy where catalytic DNA that are selective for competing metal ions are discarded in the in vitro selection processes. By labeling the resulting catalytic DNA with a fluorophore/quencher pair, we have made a new class of metal ion fluorescent sensors that are the first examples of catalytic DNA biosensors for metal ions. The sensors combine the high selectivity of catalytic DNA with the high sensitivity of fluorescent detection, and can be applied to the quantitative detection of metal ions over a wide concentration range and with high selectivity. The use of DNA sensors in detection and quantification of lead ions in environmental samples such as water from Lake Michigan has been demonstrated. DNA is stable, cost-effective, environmentally benign, and easily adaptable to optical fiber and microarray technology for device manufacture. Thus, the DNA sensors explained here hold great promise for on-site and real-time monitoring of metal ions in the fields of environmental monitoring, developmental biology, clinical toxicology, wastewater treatment, and industrial process monitoring.
45 | Juewen Liu, Yi Lu
"FRET Study of a Trifluorophore-Labeled DNAzyme"
Journal of the American Chemical Society 124, 15208-15216 (2002) [PDF] [doi]

A fluorescence resonance energy transfer (FRET) study of biomolecules typically employs two fluorophores. The increasing number of branches and complexity of biomolecules call for simultaneously monitoring structures and dynamics of several branches in a single system. Furthermore, despite recent studies that show DNAzymes can be a stable and cost-effective alternative to protein and ribozymes for pharmaceutical and biotechnological applications, no FRET study of DNAzymes has been reported. Here, we describe the FRET study of a trifluorophore-labeled “8-17” DNAzyme, in which each of the three branches is labeled with a different fluorophore. From the study, we found that the (ratio)A method that has been commonly used in dual-fluorophore-labeled systems is also applicable to trifluorophore-labeled systems. However, while both FRET efficiency and fluorophore-to-fluorophore distance can be used to measure FRET in dual-fluorophore-labeled systems, only the average distance should be used in trifluorophore-labeled systems. The ability to monitor all three branches in a single system allowed us to reveal new metal-ion-dependent conformational changes in the DNAzyme. The trifluorophore-labeled “8-17” DNAzyme has been found to adopt a two-step folding process in the presence of Zn2+. Each step is induced by one Zn2+ binding, with apparent dissociation constants of 19 μM and 260 μM for binding the first and second Zn2+, respectively. The trifluorophore FRET results are verified by a dual-labeled control experiment. The results demonstrated that the trifluorophore-labeled system is simple and yet powerful in studying complicated biomolecular structure and dynamics and is capable of revealing new sophisticated structural changes that may have functional implications.
44 | Geoffrey A. Holloway, Caroline Pavot, Stephen A. Scaringe, Yi Lu, Thomas B. Rauchfuss
"An Organometallic Route to Oligonucleotides Containing Phosphoroselenoate"
ChemBioChem 3, 1061-1065 (2002) [PDF] [doi]

Unlike the widespread use of phosphorothioates in nucleic acid chemistry, complementary research on phosphoroselenoates has been severely limited. Previous routes to DNA and RNA that contain phosphoroselenoates employ elemental Se and KSeCN as Se transfer agents, although these reagents suffer from low or unselective reactivity. The metastability of the P&bond;Se bond demands soluble, selective Se transfer reagents. The organometallic reagent (iPrC5H4)2TiSe5 satisfies these criteria, as we demonstrate by the synthesis of phosphoroselenoate derivatives of mono- and oligonucleotides of DNA and a dinucleotide of RNA. The new general method is compatible with high-throughput phosphoramidate oligonucleotide synthesis, which allows for the preparation of site-specifically labeled oligonucleotides. A 31P NMR spectroscopy study shows that the phosphoroselenoate of (5prime)-d(GGAATGTCSeTGTCG)-(3prime) selectively binds to soft Cd2+ ions but not Mg2+ ions.
43 | Boris Epel, Claire S. Slutter, Frank Neese, Peter M. H. Kroneck, Walter G. Zumft, Israel Pecht, Ole Farver, Yi Lu, Daniella Goldfarb
"Electron-Mediating CuA Centers in Proteins: A Comparative High Field 1H ENDOR Study"
Journal of the American Chemical Society 124, 8152-8162 (2002) [PDF] [doi]

High field (W-band, 95 GHz) pulsed electron-nuclear double resonance (ENDOR) measurements were carried out on a number of proteins that contain the mixed-valence, binuclear electron-mediating CuA center. These include nitrous oxide reductase (N2OR), the recombinant water-soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase (COX) ba3 (M160T9), its M160QT0 mutant, where the weak axial methionine ligand has been replaced by a glutamine, and the engineered purple azurin (purpAz). The three-dimensional (3-D) structures of these proteins, apart from the mutant, are known. The EPR spectra of all samples showed the presence of a mononuclear Cu(II) impurity with EPR characteristics of a type II copper. At W-band, the g features of this center and of CuA are well resolved, thus allowing us to obtain a clean CuA ENDOR spectrum. The latter consists of two types of ENDOR signals. The first includes the signals of the four strongly coupled cysteine Beta-protons, with isotropic hyperfine couplings, Aiso, in the 7-15 MHz range. The second group consists of weakly coupled protons with a primarily anisotropic character with Azz < 3 MHz. Orientation selective ENDOR spectra were collected for N2OR, M160QT0, and purpAz, and simulations of the cysteine Beta-protons signals provided their isotropic and anisotropic hyperfine interactions. A linear correlation with a negative slope was found between the maximum Aiso value of the Beta-protons and the copper hyperfine interaction. Comparison of the best-fit anisotropic hyperfine parameters with those calculated from dipolar interactions extracted from the available 3-D structures sets limit to the sulfur spin densities. Similarly, the small coupling spectral region was simulated on the basis of the 3-D structures and compared with the experimental spectra. It was found that the width of the powder patterns of the weakly coupled protons recorded at g is mainly determined by the histidine HE1 protons. Furthermore, the splitting in the outer wings of these powder patterns indicates differences in the positions of the imidazole rings relative to the Cu2S2 core. Comparison of the spectral features of the weakly coupled protons of M160QT0 with those of the other investigated proteins shows that they are very similar to those of purpAz, where the CuA center is the most symmetric, but the copper spin density and the HE1-Cu distances are somewhat smaller. All proteins show the presence of a proton with a significantly negative Aiso value which is assigned to an amide proton of one of the cysteines. The simulations of both strongly and weakly coupled protons, along with the known copper hyperfine couplings, were used to estimate and compare the spin density distribution in the various CuA centers. The largest sulfur spin density was found in M160T9, and the lowest was found in purpAz. In addition, using the relation between the Aiso values of the four cysteine Beta-protons and the H-C-S-S dihedral angles, the relative contribution of the hyperconjugation mechanism to Aiso was determined. The largest contribution was found for M160T9, and the lowest was found for purpAz. Possible correlations between the spin density distribution, structural features, and electron-transfer functionality are finally suggested.
42 | Dmitriy Lukoyanov, Steven M. Berry, Yi Lu, William E. Antholine, Charles P. Scholes
"Role of the Coordinating Histidine in Altering the Mixed Valency of CuA: An Electron Nuclear Double Resonance-Electron Paramagnetic Resonance Investigation"
Biophysical Journal 82, 2758-2766 (2002) [PDF] [doi]

The binuclear CuA site engineered into Pseudomonas aeruginosa azurin has provided a CuA-azurin with a well-defined crystal structure and a CuSSCu core having two equatorial histidine ligands, His120 and His46. The mutations His120Asn and His120Gly were made at the equatorial His120 ligand to understand the histidine-related modulation to CuA, notably to the valence delocalization over the CuSSCu core. For these His120 mutants Q-band electron nuclear double resonance (ENDOR) and multifrequency electron paramagnetic resonance (EPR) (X, C, and S-band), all carried out under comparable cryogenic conditions, have provided markedly different electronic measures of the mutation-induced change. Q-band ENDOR of cysteine C[beta] protons, of weakly dipolar-coupled protons, and of the remaining His46 nitrogen ligand provided hyperfine couplings that were like those of other binuclear mixed-valence CuA systems and were essentially unperturbed by the mutation at His120. The ENDOR findings imply that the CuA core electronic structure remains unchanged by the His120 mutation. On the other hand, multifrequency EPR indicated that the H120N and H120G mutations had changed the EPR hyperfine signature from a 7-line to a 4-line pattern, consistent with trapped-valence, Type 1 mononuclear copper. The multifrequency EPR data imply that the electron spin had become localized on one copper by the His120 mutation. To reconcile the EPR and ENDOR findings for the His120 mutants requires that either: if valence localization to one copper has occurred, the spin density on the cysteine sulfurs and the remaining histidine (His46) must remain as it was for a delocalized binuclear CuA center, or if valence delocalization persists, the hyperfine coupling for one copper must markedly diminish while the overall spin distribution on the CuSSCu core is preserved.
41 | Yi Lu
"New Transition-Metal-Dependent DNAzymes as Efficient Endonucleases and as Selective Metal Biosensors"
Chemistry - A European Journal 8, 4588-4596 (2002) [PDF] [doi]

Like proteins and RNA molecules, many DNA molecules have now been shown to catalyze a variety of reactions and are thus called DNAzymes. With limited building blocks, DNAzymes need to recruit other cofactors in order to match other enzymes in terms of reaction diversity and catalytic efficiency. Several unique properties make transition-metal ions an ideal choice of cofactor for DNAzymes. Indeed, new DNAzymes that bind transition-metal ions with high affinity and selectivity have been obtained through the use of a powerful combinatorial biology tool called in vitro selection. This accomplishment now makes it possible to obtain different classes of metallo-DNAzymes in the laboratory within a short period of time. It also offers a rare opportunity to compare and contrast structural and functional properties of metal-binding sites in proteins and in DNAzymes. The resulting transition-metal-dependent DNAzymes have displayed high activity toward cleavage of DNA and RNA and thus hold promise for their biochemical and pharmaceutical applications. Finally, the use of DNAzymes as a new class of highly sensitive and selective biosensors for metal ions has been demonstrated recently.
40 | Steven M. Berry, Matt D. Gieselman, Mark J. Nilges, Wilfred A. van der Donk, Yi Lu
"An Engineered Azurin Variant Containing a Selenocysteine Copper Ligand"
Journal of the American Chemical Society 124, 2084-2085 (2002) [PDF] [doi]

Modulating the properties of proteins through de novo design or redesign of existing proteins has been a longstanding goal in protein chemistry. Over the past two decades, site-directed mutagenesis has been a powerful tool to probe the role of certain residues and to fine-tune the activity of proteins. A limitation of this approach has been the accessibility of only a restricted number of functional groups through the 20 amino acids in the genetic code. The more recent technique of expressed protein ligation (EPL) provides an alternative route that allows efficient incorporation of nonnatural residues into proteins. We report here the preparation and spectroscopic characterization of an azurin variant in which a cysteine ligand to the blue copper center has been replaced by EPL with selenocysteine (Sec). This reports marks the first time that selenocysteine is artificially incorporated into the active site of a metalloprotein. The variant displays a significantly increased A|| (from 56 to 104 G) and red-shifted CT band (from 625 to 677 nm), while maintaining the general type 1 copper characteristics, including similarity in reduction potentials. This study illustrates that iso-structural substitution using EPL can fine-tune the structural and functional properties of a metal-binding site without loss of most of its characteristics. Further spectroscopic and X-ray crystallographic studies of this and other EPL variants will provide new insights into the fine-control of the structure and function of metalloproteins.
39 | Bruesehoff P.J., Li J., Augustine III A.J., Lu Y.
"Improving Metal Ion Specificity During In Vitro Selection of Catalytic DNA"
Combinatorial Chemistry & High Throughput Screening 5, 327-335 (2002) [doi]
38 | Thomas D. Pfister, Alan J. Gengenbach, Sung Syn, Yi Lu
"The Role of Redox-Active Amino Acids on Compound I Stability, Substrate Oxidation, and Protein Cross-Linking in Yeast Cytochrome c Peroxidase"
Biochemistry 40, 14942-14951 (2001) [PDF] [doi]

The role of two tryptophans (Trp51 and Trp191) and six tyrosines (Tyr36, Tyr39, Tyr42, Tyr187, Tyr229, and Tyr236) in yeast cytochrome c peroxidase (CcP) has been probed by site-directed mutagenesis. A series of sequential mutations of these redox-active amino acid residues to the corresponding, less oxidizable residues in lignin peroxidase (LiP) resulted in an increasingly more stable compound I, with rate constants for compound I decay decreasing from 57 s-1 for CcP(MI, W191F) to 7 s-1 for CcP(MI, W191F,W51F,Y187F,Y229F,Y236F,Y36F,Y39E,Y42F). These results provide experimental support for the proposal that the stability of compound I depends on the number of endogenous oxidizable amino acids in proteins. The higher stability of compound I in the variant proteins also makes it possible to observe its visible absorption spectroscopic features more clearly. The effects of the mutations on oxidation of ferrocytochrome c and 2,6-dimethoxyphenol were also examined. Since the first mutation in the series involved the change of Trp191, a residue that plays a critical role in the electron transfer pathway between CcP and cyt c, the ability to oxidize cyt c was negligible for all mutant proteins. On the other hand, the W191F mutation had little effect on the proteins' ability to oxidize 2,6-dimethoxyphenol. Instead, the W51F mutation resulted in the largest increase in the kcat/KM, from 2.1 × 102 to 5.0 × 103 M-1 s-1, yielding an efficiency that is comparable to that of manganese peroxidase (MnP). The effect in W51F mutation can be attributed to the residue's influence on the stability and thus reactivity of the ferryl oxygen of compound II, whose substrate oxidation is the rate-determining step in the reaction mechanism. Finally, out of all mutant proteins in this study, only the variant containing the Y36F, Y39E, and Y42F mutations was found to prevent covalent protein cross-links in the presence of excess hydrogen peroxide and in the absence of exogenous reductants. This finding marks the first time a CcP variant is incapable of forming protein cross-links and confirms that one of the three tyrosines must be involved in the protein cross-linking.
37 | Yi Lu, Steven M. Berry, Thomas D. Pfister
"Engineering Novel Metalloproteins: Design of Metal-Binding Sites into Native Protein Scaffolds"
Chemical Reviews 101, 3047-3080 (2001) [PDF] [doi]
36 | Yi Lu
"What's in a Name"
Chemical and Engineering News 79, (13) 241 (2001) [PDF] [news]
35 | Jeffrey A. Sigman, Xiaotang Wang, Yi Lu
"Coupled Oxidation of Heme by Myoglobin Is Mediated by Exogenous Peroxide"
Journal of the American Chemical Society 123, 6945-6946 (2001) [PDF] [doi]

34 | Serena DeBeer George, Markus Metz, Robert K. Szilagyi, Hongxin Wang, Stephen P. Cramer, Yi Lu, William B. Tolman, Britt Hedman, Keith O. Hodgson, Edward I. Solomon
"A Quantitative Description of the Ground-State Wave Function of CuA by X-ray Absorption Spectroscopy:  Comparison to Plastocyanin and Relevance to Electron Transfer"
Journal of the American Chemical Society 123, 5757-5767 (2001) [PDF] [doi]

To evaluate the importance of the electronic structure of CuA to its electron-transfer (ET) function, a quantitative description of the ground-state wave function of the mixed-valence (MV) binuclear CuA center engineered into Pseudomonas aeruginosa azurin has been developed, using a combination of S K-edge and Cu L-edge X-ray absorption spectroscopies (XAS). Parallel descriptions have been developed for a binuclear thiolate-bridged MV reference model complex ([(LiPrdacoSCu)2]+) and a homovalent (II,II) analogue ([LiPr2tacnSCu)2]2+, where LiPrdacoS and LiPr2tacnS are macrocyclic ligands with attached thiolates that bridge the Cu ions. Previous studies have qualitatively defined the ground-state wave function of CuA in terms of ligand field effects on the orbital orientation and the presence of a metal−metal bond. The studies presented here provide further evidence for a direct Cu−Cu interaction and, importantly, experimentally quantify the covalency of the ground-state wave function. The experimental results are further supported by DFT calculations. The nature of the ground-state wave function of CuA is compared to that of the well-defined blue copper site in plastocyanin, and the importance of this wave function to the lower reorganization energy and ET function of CuA is discussed. This wave function incorporates anisotropic covalency into the intra- and intermolecular ET pathways in cytochrome c oxidase. Thus, the high covalency of the Cys−Cu bond allows a path through this ligand to become competitive with a shorter His path in the intramolecular ET from CuA to heme a and is particularly important for activating the intermolecular ET path from heme c to CuA.
33 | Paola Turano, Yi Lu
"Iron in Heme and Related Proteins"
in "Handbook on Metalloproteins", Ivano Bertini, Helmut Sigel, Astrid Sigel, eds.; Marcel Dekker, Inc.; pp. 269-356 (2001)
32 | Gengenbach Alan, Wang Xiaotang, Lu Yi
"Designing a Manganese Peroxidase"
in "Oxidative Delignification Chemistry", ACS Symposium Series, Vol. 785, Dimitris S. Argyropoulos, ed.; ACS: Washington, D.C.; pp. 487-500 (2001) [PDF] [doi]

Manganese peroxidase (MnP) from the white rot fungus Phanerochaete chrysosporium plays a vital role in lignin degradation. MnP functions by oxidizing Mn(II) to Mn(III) which serves as a diffusible oxidant. To test the current understanding of the structure and function of MnP and to find an alternative catalyst for oxidative delignification, we employed a new approach involving the design and engineering of a MnP using cytochrome c peroxidase (CcP) from bakers' yeast. Based on structural comparisons and computer modeling, we created a CcP mutant (MnCcP) that binds Mn(II) in a manner similar to the native enzyme and showed that the incorporation of the Mn(II)-binding site facilitates Mn(II) oxidation. Further mutations of key active site residues (W191 and W51) in MnCcP to the corresponding Phe in MnP conferred even more MnP activity of our protein model. The two mutations do not contribute equally to the activity increase. A much larger increase arises from the W51F mutation because W51 stabilizes compound II. Since the reaction of compound II with substrate is rate-limiting, a more reactive compound II increases MnP activity. Thus our approach is also capable of offering new insight into the structure↔function relationships of MnP and CcP.
31 | Jing Li, Yi Lu
"A Highly Sensitive and Selective Catalytic DNA Biosensor for Lead Ions"
Journal of the American Chemical Society 122, 10466-10467 (2000) [PDF] [doi]

30 | Michael T. Hay, Yi Lu
"Metal-binding properties of an engineered purple CuA center in azurin"
Journal of Biological Inorganic Chemistry 5, 699-712 (2000) [PDF] [doi]

A CuA center engineered into Pseudomonas aeruginosa azurin was studied by metal substitution. Metal-binding properties were determined by electronic absorption (UV-vis) and electrospray ionization mass spectrometry (ESI-MS). The metal-binding site readily binds thiophilic metal ions, such as Hg(II), Ag(I), Cu(I), Cd(II), and Au(I). Harder metal ions, like Co(II), bind to apo-CuA-azurin only under basic conditions (pH 9.1-9.2). The results obtained from these studies indicate that two factors influence metal binding in CuA azurin: (1) the site favors metal combinations which produce an overall +3 charge, and (2) the site binds soft, thiophilic metal ions. The results demonstrate the remarkable ability of the CuA center to maintain valence delocalization of its native metal ions and to ensure redox accessibility of only one of the two redox couples (i.e., [Cu(1.5)&\183;&\183;&\183;Cu(1.5)]&\85[Cu(I)&\183;&\183;&\183;Cu(I)]) under physiological conditions. These findings may lead to the preparation of new metal ion derivatives and can serve as a basis for understanding this efficient electron transfer center.
29 | Jeffrey A. Sigman, Brian C. Kwok, Yi Lu
"From Myoglobin to Heme-Copper Oxidase:  Design and Engineering of a CuB Center into Sperm Whale Myoglobin"
Journal of the American Chemical Society 122, 8192-8196 (2000) [PDF] [doi]

Myoglobins (Mb) are small globular heme proteins that serve as O2 carriers. Heme-copper oxidases (HCOs) are large membrane-bound proteins that catalyze proton-coupled reduction of O2 to water. Mb contains a single heme center, while HCOs contain a high-spin heme-CuB dinuclear center, a low-spin heme center, and in certain subclasses of HCO enzymes such as cytochrome c oxidases (CcO) a dinuclear copper center called CuA. While Mb is one of the most well-characterized proteins, many questions about the structure and function of HCOs, such as the role of the CuB center, the origin of spin coupling between CuB and heme, and the exact nature of the reaction intermediates, remain to be fully understood. We report here the design and engineering of a copper-binding site in sperm whale myoglobin (swMb) based on structural comparison and computer modeling of swMb and CcO. UV−vis studies of the resting state of the designed protein swMb((L29H, F43H) (called CuBMb) suggest that a single copper-binding site is created in swMb. UV−vis, elemental analysis, and EPR studies of the cyanide-bound CuBMb indicate that a spin-coupled, CN--bridged CuB-heme center is formed in the designed model protein, as in the native HCOs. Parallel spectroscopic studies with Zn(II) in the place of Cu(II) further support the conclusion. The study also reveals that the presence of Cu(II) and Ag(I) (as a Cu(I) mimic) increased the affinity of heme for diatomic ligands such as CN- and O2. This study shows that it is possible to design and engineer metal-binding sites in proteins with little sequence and structural homology. The resulting designed protein, free from other chromophores, is more amendable to biochemical and biophysical studies. Spectroscopy studies of the designed protein indicate that the CuB center plays an important role in HCO structure and function.
28 | Jing Li, Wenchao Zheng, Angela H. Kwon, Yi Lu
"In vitro selection and characterization of a highly efficient Zn(II)-dependent RNA-cleaving deoxyribozyme"
Nucl. Acids Res. 28, 481-488 (2000) [PDF] [doi]

A group of highly efficient Zn(II)-dependent RNA-cleaving deoxyribozymes has been obtained through in vitro selection. They share a common motif with the 8-17' deoxyribozyme isolated under different conditions, including different design of the random pool and metal ion cofactor. We found that this commonly selected motif can efficiently cleave both RNA and DNA/RNA chimeric substrates. It can cleave any substrate containing rNG (where rN is any ribo-nucleotide base and G can be either ribo- or deoxy-ribo-G). The pH profile and reaction products of this deoxyribozyme are similar to those reported for hammerhead ribozyme. This deoxyribozyme has higher activity in the presence of transition metal ions compared to alkaline earth metal ions. At saturating concentrations of Zn2+, the cleavage rate is 1.35 min-1 at pH 6.0; based on pH profile this rate is estimated to be at least  30 times faster at pH 7.5, where most assays of Mg2+-dependent DNA and RNA enzymes are carried out. This work represents a comprehensive characterization of a nucleic acid-based endonuclease that prefers transition metal ions to alkaline earth metal ions. The results demonstrate that nucleic acid enzymes are capable of binding transition metal ions such as Zn2+ with high affinity, and the resulting enzymes are more efficient at RNA cleavage than most Mg2+-dependent nucleic acid enzymes under similar conditions.
27 | Steven M. Berry, Xiaotang Wang, Yi Lu
"Ligand replacement study at the His120 site of purple CuA azurin"
Journal of Inorganic Biochemistry 78, 89 - 95 (2000) [PDF] [doi]

The CuA center is a dinuclear Cu2S2(Cys) electron transfer center found in cytochrome c oxidase and nitrous oxide reductase. In a previous investigation of the equatorial histidine ligands' effect on the reduction potential, electron transfer and spectroscopic properties of the CuA center, His120 in the engineered CuA azurin was mutated to Asn, Asp, and Ala. The identical absorption and EPR spectra of these mutants indicate that a common ligand is bound to the copper center. To identify this replacement ligand, the His120Gly CuA azurin mutant was constructed and purified. Absorption and X-band EPR spectra show that His120Gly is similar to the other His120X (X=Asn, Asp, Ala) mutant proteins. Titrations with chloride, imidazole, and azide suggest that the replacement ligand is not exchangeable with exogenous ligands. The possibility of an internal amino acid acting as the replacement ligand for His120 in the His120X mutant proteins was investigated by analyzing the CuA azurin crystal structure and then converting the likely internal ligand, Asn119, to Asp, Ser, or Ala in the His120Gly mutant. The double mutants H120G/Asn119X (X=Asp, Ser, or Ala) displayed UV-Vis absorption and EPR spectra that are identical to His120Gly and the other His120X mutants, indicating that Asn119 is not the internal ligand replacing His120 in the His120X mutant proteins. These results demonstrate the remarkable stability of the dinuclear His120 mutants of CuA azurin.
26 | Lynette Cunningham, Jing Li, Yi Lu
"Looking for the Needles in a Haystack: Spectroscopic Characterization of Catalytic Metal Binding Site in Nucleic Acid Enzymes"
in "Symposium on RNA Biology III. RNA, Tool and Target", Nucleic acids symposium series, pp. 70-72 (1999)
25 | Jeffrey A. Sigman, Brian C. Kwok, Alan Gengenbach, Yi Lu
"Design and Creation of a Cu(II)-Binding Site in Cytochrome c Peroxidase that Mimics the CuB-heme Center in Terminal Oxidases"
Journal of the American Chemical Society 121, 8949-8950 (1999) [doi]

24 | Alan Gengenbach, Sung Syn, Xiaotang Wang, Yi Lu
"Redesign of Cytochrome c Peroxidase into a Manganese Peroxidase: Role of Tryptophans in Peroxidase Activity†"
Biochemistry 38, 11425-11432 (1999) [PDF] [doi]

Trp191Phe and Trp51Phe mutations have been introduced into an engineered cytochrome c peroxidase (CcP) containing a Mn(II)-binding site reported previously (MnCcP; see Yeung, B. K.-S., et al. (1997) Chem. Biol. 5, 215221). The goal of the present study is to elucidate the role of tryptophans in peroxidase activity since CcP contains both Trp51 and Trp191 while manganese peroxidase (MnP) contains phenylalanine residues at the corresponding positions. The presence of Trp191 in CcP allows formation of a unique high-valent intermediate containing a ferryl oxo and tryptophan radical called compound I`. The absence of a tryptophan residue at this position in MnP is the main reason for the formation of an intermediate called compound I which contains a ferryl oxo and porphyrin pi-cation radical. In this study, we showed that introduction of the Trp191Phe mutation to MnCcP did not improve MnP activity (specific activity: MnCcP, 0.750 mumol min-1 mg-1; MnCcP(W191F), 0.560 mumol min-1 mg-1. kcat/Km: MnCcP, 0.0517 s-1 mM-1; MnCcP(W191F), 0.0568 s-1 mM-1) despite the fact that introduction of the same mutation to WTCcP caused the formation of a transient compound I (decay rate, 60 s-1). However, introducing both the Trp191Phe and Trp51Phe mutations not only resulted in a longer lived compound I in WTCcP (decay rate, 18 s-1), but also significantly improved MnP activity in MnCcP (MnCcP(W51F, W191F): specific activity, 8.0 mumol min-1 mg-1; kcat/Km, 0.599 s-1 mM-1). The increase in activity can be attributed to the Trp51Phe mutation since MnCcP(W51F) showed significantly increased MnP activity relative to MnCcP (specific activity, 3.2 mumol min-1 mg-1; kcat/Km, 0.325 s-1 mM-1). As with MnP, the activity of MnCcP(W51F, W191F) was found to increase with decreasing pH. Our results demonstrate that, while the Trp191Phe and Trp51Phe mutations both play important roles in stabilizing compound I, only the Trp51Phe mutation contributes significantly to increasing the MnP activity because this mutation increases the reactivity of compound II, whose oxidation of Mn(II) is the rate-determining step in the reaction mechanism.
23 | Jeffrey A. Sigman, Alycen E. Pond, John H. Dawson, Yi Lu
"Engineering Cytochrome c Peroxidase into Cytochrome P450: A Proximal Effect on Heme−Thiolate Ligation†"
Biochemistry 38, 11122-11129 (1999) [PDF] [doi]

In an effort to investigate factors required to stabilize hemethiolate ligation, key structural components necessary to convert cytochrome c peroxidase (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been evaluated and the H175C/D235L CcP double mutant has been engineered. The UVvisible absorption, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra for the double mutant at pH 8.0 are reported herein. The close similarity between the spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric state of the double mutant with all three techniques support the conclusion that the latter has a pentacoordinate, high-spin heme with thiolate ligation. Previous efforts to prepare a thiolate-ligated mutant of CcP with the H175C single mutant led to Cys oxidation to cysteic acid [Choudhury et al. (1994) J. Biol. Chem. 267, 2565625659]. Therefore it is concluded that changing the proximal Asp235 residue to Leu is critical in forming a stable hemethiolate ligation in the resting state of the enzyme. To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin complexes have also been prepared. Addition of the neutral ligand imidazole or of the anionic ligand cyanide results in formation of hexacoordinate adducts that retain thiolate ligation as determined by spectral comparison to the analogous derivatives of ferric P450cam. The stability of these complexes and their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP double mutant as a model for ferric P450 enzymes. This study marks the first time a stable cyanoferric complex of a model P450 has been made and demonstrates the importance of the environment around the primary coordination ligands in stabilizing metalligand ligation.
22 | Xiaotang Wang, Steven M. Berry, Yaomin Xia, Yi Lu
"The Role of Histidine Ligands in the Structure of Purple CuA Azurin"
Journal of the American Chemical Society 121, 7449-7450 (1999) [PDF] [doi]

21 | Xiaotang Wang, Yi Lu
"Proton NMR Investigation of the Heme Active Site Structure of an Engineered Cytochrome c Peroxidase that Mimics Manganese Peroxidase†"
Biochemistry 38, 9146-9157 (1999) [PDF] [doi]

The heme active site structure of an engineered cytochrome c peroxidase [MnCcP; see Yeung, B. K., et al. (1997) Chem. Biol. 4, 215221] that closely mimics manganese peroxidase (MnP) has been characterized by both one- and two-dimensional NMR spectroscopy. All hyperfine-shifted resonances from the heme pocket as well as resonances from catalytically relevant amino acid residues in the congested diamagnetic envelope have been assigned. From the NMR spectral assignment and the line broadening pattern of specific protons in NOESY spectra of MnCcP, the location of the engineered Mn(II) center is firmly identified. Furthermore, we found that the creation of the Mn(II)-binding site in CcP resulted in no detectable structural changes on the distal heme pocket of the protein. However, notable structural changes are observed at the proximal side of the heme cavity. Both CepsilonH shift of the proximal histidine and 15N shift of the bound C15N- suggest a weaker heme Fe(III)N(His) bond in MnCcP compared to WtCcP. Our results indicate that the engineered Mn(II)-binding site in CcP resulted in not only a similar Mn(II)-binding affinity and improved MnP activity, but also weakened the Fe(III)N(His) bond strength of the template protein CcP so that its bond strength is similar to that of the target protein MnP. The results presented here help elucidate the impact of designing a metal-binding site on both the local and global structure of the enzyme, and provide a structural basis for engineering the next generation of MnCcP that mimics MnP more closely.
20 | Xiaotang Wang, Marjorie C. Ang, Yi Lu
"Kinetics of Copper Incorporation into an Engineered Purple Azurin"
Journal of the American Chemical Society 121, 2947-2948 (1999) [PDF] [doi]

19 | Howard Robinson, Marjorie C. Ang, Yi-Gui Gao, Michael T. Hay, Yi Lu, Andrew H.-J. Wang
"Structural Basis of Electron Transfer Modulation in the Purple CuA Center"
Biochemistry 38, 5677-5683 (1999) [PDF] [doi]

The X-ray structure of an engineered purple CuA center in azurin from Pseudomonas aeruginosa has been determined and refined at 1.65 resolution. Two independent purple CuA azurin molecules are in the asymmetric unit of a new P21 crystal, and they have nearly identical conformations (rmsd of 0.27 for backbone atoms). The purple CuA azurin was produced by the loop-engineering strategy, and the resulting overall structure is unperturbed. The insertion of a slightly larger Cu-binding loop into azurin causes the two structural domains of azurin to move away from each other. The high-resolution structure reveals the detailed environment of the delocalized mixed-valence [Cu(1.5)Cu(1.5)] binuclear purple CuA center, which serves as a useful reference model for other native proteins, and provides a firm basis for understanding results from spectroscopic and functional studies of this class of copper center in biology. The two independent Cu-Cu distances of 2.42 and 2.35 (with respective concomitant adjustments of ligand-Cu distances) are consistent with that (2.39 ) obtained from X-ray absorption spectroscopy with the same molecule, and are among the shortest Cu-Cu bonds observed to date in proteins or inorganic complexes. A comparison of the purple CuA azurin structure with those of other CuA centers reveals an important relationship between the angular position of the two His imidazole rings with respect to the Cu2S2(Cys) core plane and the distance between the Cu and the axial ligand. This relationship strongly suggests that the fine structural variation of different CuA centers can be correlated with the angular positions of the two histidine rings because, from these positions, one can predict the relative axial ligand interactions, which are responsible for modulating the Cu-Cu distance and the electron transfer properties of the CuA centers.
18 | Ole Farver, Yi Lu, Majorie C. Ang, Israel Pecht
"Enhanced rate of intramolecular electron transfer in an engineered purple CuA azurin"
Proceedings of the National Academy of Sciences of the United States of America 96, 899-902 (1999) [PDF] [doi]

The recent expression of an azurin mutant where the blue type 1 copper site is replaced by the purple CuA site of Paracoccus denitrificans cytochrome c oxidase has yielded an optimal system for examining the unique electron mediation properties of the binuclear CuA center, because both type 1 and CuA centers are placed in the same location in the protein while all other structural elements remain the same. Long-range electron transfer is induced between the disulfide radical anion, produced pulse radiolytically, and the oxidized binuclear CuA center in the purple azurin mutant. The rate constant of this intramolecular process, k ET = 650 ± 60 s−1 at 298 K and pH 5.1, is almost 3-fold faster than for the same process in the wild-type single blue copper azurin from Pseudomonas aeruginosa (250 ± 20 s−1), in spite of a smaller driving force (0.69 eV for purple CuA azurin vs. 0.76 eV for blue copper azurin). The reorganization energy of the CuA center is calculated to be 0.4 eV, which is only 50% of that found for the wild-type azurin. These results represent a direct comparison of electron transfer properties of the blue and purple CuA sites in the same protein framework and provide support for the notion that the binuclear purple CuA center is a more efficient electron transfer agent than the blue single copper center because reactivity of the former involves a lower reorganization energy.
17 | Tu H. Nguyen, Lynette A. Cunningham, Kendra M. Hammond, Yi Lu
"High-Resolution Preparative-Scale Purification of RNA Using the Prep Cell,"
Analytical Biochemistry 269, 216 - 218 (1999) [PDF] [doi]
16 | Lynette A. Cunningham, Jing Li, Yi Lu
"Spectroscopic Evidence for Inner-Sphere Coordination of Metal Ions to the Active Site of a Hammerhead Ribozyme"
Journal of the American Chemical Society 120, 4518-4519 (1998) [PDF] [doi]
15 | Daniel R. Gamelin, David W. Randall, Michael T. Hay, Robert P. Houser, Ton C. Mulder, Gerard W. Canters, Simon de Vries, William B. Tolman, Yi Lu, Edward I. Solomon
"Spectroscopy of Mixed-Valence CuA-Type Centers: Ligand-Field Control of Ground-State Properties Related to Electron Transfer"
Journal of the American Chemical Society 120, 5246-5263 (1998) [PDF] [doi]

Using a combination of electronic spectroscopies, electronic structural descriptions have been developed for a series of binuclear CuA-type centers in Bacillus subtilis CcO and engineered into the blue copper proteins Pseudomonas aeruginosa azurin and Thiobacillus versutus amicyanin. Parallel descriptions are developed for two structurally characterized mixed-valence (MV) and homovalent (II,II) synthetic copper thiolate dimers. Assignment of the excited-state spectral features allows the electronic structures of CuA and the MV model to be understood and compared in relation to their copper coordination environments. These electronic structural descriptions are supported by SCF-Xα-SW MO calculations, which are used to test systematically the effects of major structural perturbations linking the MV model geometry to that of CuA. It is determined that both Cu−Cu compression and removal of the axial ligands are critical determinants of the orbital ground state in these dimers. The weakened axial interactions in CuA appear to parallel the mechanism for protein control of electron transfer (ET) function observed in blue copper centers. The major geometric and electronic features of CuA, including metal−ligand covalency, redox potentials, reorganization energies, valence delocalization, and the weakened axial bonding interactions, are discussed in relation to its ET function, and specific potential ET pathways are identified and compared.
14 | Blackburn N. J., Ralle M., Sanders D., Fee J. A., de Vries S., Houser R. P., Tolman W. B., Hay M. T., Lu Y.
"XAS Studies on the CuA Centers of Heme-Copper Oxidases and Loop-Directed Mutants of Azurin: Implications for Redox Reactivity"
in "Spectroscopic Methods in Bioinorganic Chemistry", ACS Symposium Series, Vol. 692, Edward I. Solomon, Keith O. Hodgson, eds.; ACS: Washington, D.C.; pp. 241-259 (1998) [PDF] [doi]

The heme-copper oxidases (historically termed cytochrome oxidases) are ubiquitous in both prokaryotic and eukaryotic aerobic organisms. They function to catalyze the 4-proton, 4-electron reduction of dioxygen to water and transduce the energy made available from O-O bond cleavage to the translocation of an additional 4 protons across the cytoplasmic or mitochondrial membrane. The energy released is stored as an electrochemical gradient and utilized to drive ATP synthesis in the process known as oxidative phosphorylation. For these reasons, heme-copper oxidases are arguably the most important enzymes in mammalian cells, providing the energy for most metabolic processes and consuming 80-90% of the oxygen we breathe. The catalytic activity of the heme copper oxidases resides in subunit I which is a membrane-spanning polypeptide, containing a dinuclear high-spin heme-CuB dioxygen binding site and a low-spin heme center. A second subunit (II) contains the binding site for the electron donor. A number of differing classes
13 | Michael T. Hay, Marjorie C. Ang, Daniel R. Gamelin, Edward I. Solomon, William E. Antholine, Martina Ralle, Ninian J. Blackburn, Priscilla D. Massey, Xiaotang Wang, Angela H. Kwon, Yi Lu
"Spectroscopic Characterization of an Engineered Purple CuA Center in Azurin"
Inorganic Chemistry 37, 191-198 (1998) [PDF] [archived PDF] [doi]

The mixed-valent [Cu(1.5)...Cu(1.5)] purple CuA center, a new class of copper centers in biology, has been engineered into a classic type 1 blue copper azurin using loop-directed mutagenesis. Comprehensive spectroscopic study using UVvis, ES-MS, MCD, multifrequency EPR, and XAS showed striking similarity between the engineered CuA center in azurin and other native and engineered CuA centers, demonstrating the close structural relationship between the two copper centers and the power of redesigning metal-binding sites in elucidating the structure and function of metalloproteins.
12 | Yi Lu, Joan S Valentine
"Engineering metal-binding sites in proteins"
Current Opinion in Structural Biology 7, 495 - 500 (1997) [archived PDF] [doi]

Metal-binding sites have been engineered into both de novo designed and naturally occurring proteins. Although the redesign of existing metal-binding sites in naturally occurring proteins still offers the most promise for a successful design, the more challenging goal of engineering metal-binding sites in de novo designed proteins and peptides is being achieved with increasing frequency. Creating new metal-binding sites in naturally occurring proteins combines the strength of both approaches. Currently, all three approaches are being used effectively in elucidating the structure and function of naturally occurring metalloproteins.
11 | Bryan K.S. Yeung, Xiaotang Wang, Jeffrey A. Sigman, Peter A. Petillo, Yi Lu
"Construction and characterization of a manganese-binding site in cytochrome c peroxidase: towards a novel manganese peroxidase"
Chemistry & Biology 4, 215-221 (1997) [PDF] [archived PDF] [doi]

Background: Manganese-binding sites are found in several heme peroxidases, namely manganese peroxidase (MnP), chloroperoxidase, and the cationic isozyme of peanut peroxidase. The Mn-binding site in MnP is of particular interest. Oxidation of Mn(II) to Mn(III) is a key step in the biodegradation of lignin, a complex phenylpropanoid polymer, as well as of many aromatic pollutants. Cytochrome c peroxidase (CcP), which is structurally homologous to MnP despite a poor sequence homology, does not bind manganese. Thus, engineering a Mn-binding site into CcP will allow us to elucidate principles behind designing metal-binding sites in proteins, to understand the structure and function of this class of Mn-binding centers, and to prepare novel enzymes that can degrade both lignin and other xenobiotic compounds. Results: Based on a comparison of the crystal structures of CcP and MnP, a site-directed triple mutant (GIy41 Glu, Val45 - GIu, His181 Asp) of residues near the putative Mn-binding site in CcP was prepared and purified to homogeneity. Titrating MnSO4 into freshly prepared mutant CcP resulted in electronic absorption spectral changes similar to those observed in MnP. The calculated apparent dissociation constant and the stoichiometry of Mn-binding of CcP were also similar to MnP. Titration with MnSO4 resulted in the disappearance of specific paramagnetically shifted nuclear magnetic resonance spectroscopy signals assigned to residues close to the putative Mn-binding site in the mutant CcP. None of the spectral features were observed in wild-type CcP. In addition, the triple mutant was capable of oxidizing Mn(II) at least five times more efficiently than the native CcP. Conclusions: A Mn-binding site has been created in CcP and based on our spectroscopic studies the designed Mn-binding site is similar to the Mn-binding site in MnP. The results provide a basis for understanding the structure and function of the Mn-binding site and its role in different heme peroxidases.
10 | Lynette Cunningham, Kirk Kittikamron, Yi Lu
"Preparative-Scale Purification of RNA Using an Efficient Method Which Combines Gel Electrophoresis and Column Chromatography"
Nucl. Acids Res. 24, 3647-3648 (1996) [PDF] [archived PDF] [doi]

Here we describe a reliable method for purifying large amounts of RNA of any sequence and length with comparable efficiency and resolution of gel electrophoresis and with capacity approaching that of column chromatography. The RNA mixture of interest is separated on a cylindrical denaturing polyacrylamide gel, eluted by a peristaltic pump, detected by a UV-vis detector, and collected by a fraction collector. Using this method, we were able to separate one third of a 100 ml in vitro transcribed 34mer hammerhead ribozyme ([ ]6.2 mg) in a single run. The entire 100 ml transcribed RNA ([ ]18.5 mg) was separated after consecutive runs using one single gel preparation.
9 | Michael T. Hay, Richard M. Milberg, Yi Lu
"Preparation and Characterization of Mercury and Silver Derivatives of an Engineered Purple Copper Center in Azurin"
Journal of the American Chemical Society 118, 11976-11977 (1996) [PDF] [archived PDF] [doi]
8 | M Hay, J H Richards, Y Lu
"Construction and characterization of an azurin analog for the purple copper site in cytochrome c oxidase"
Proceedings of the National Academy of Sciences of the United States of America 93, 461-464 (1996) [PDF] [archived PDF]

A protein analog of a purple copper center has been constructed from a recombinant blue copper protein (Pseudomonas aeruginosa azurin) by replacing the loop containing the three ligands to the blue copper center with the corresponding loop of the CuA center in cytochrome c oxidase (COX) from Paracoccus denitrificans. The electronic absorption in the UV and visible region (UV-vis) and electron paramagnetic resonance (EPR) spectra of this analog are remarkably similar to those of the native CuA center in COX from Paracoccus denitrificans. The above spectra can be obtained upon addition of a mixture of Cu2+ and Cu+. Addition of Cu2+ only results in a UV-vis spectrum consisting of absorptions from both a purple copper center and a blue copper center. This spectrum can be converted to the spectrum of a pure purple copper by a prolonged incubation in the air, or by addition of excess ascorbate. The azurin mutant reported here is an example of an engineered purple copper center with the A480/A530 ratio greater than 1 and with no detectable hyperfines, similar to those of the CuA sites in COX of bovine heart and of Paracoccus denitrificans.
7 | Colin R. Andrew, Pekka Lappalainen, Matti Saraste, Michael T. Hay, Yi Lu, Christopher Dennison, Gerard W. Canters, James A. Fee, Nobuhumi Nakamura, Joann Sanders-Loehr
"Engineered Cupredoxins and Bacterial Cytochrome c Oxidases Have Similar CuA Sites: Evidence from Resonance Raman Spectroscopy"
Journal of the American Chemical Society 117, 10759-10760 (1995) [PDF] [archived PDF] [doi]
Postdoctoral Work
6 | Y Lu, D R Casimiro, K L Bren, J H Richards, H B Gray
"Structurally engineered cytochromes with unusual ligand-binding properties: expression of Saccharomyces cerevisiae Met-80-->Ala iso-1-cytochrome c"
Proceedings of the National Academy of Sciences of the United States of America 90, 11456-11459 (1993) [PDF] [archived PDF]

A strategy has been developed to express and purify a recombinant, nonfunctional axial-ligand mutant of iso-1-cytochrome c (Met-80-->Ala) in Saccharomyces cerevisiae in quantities necessary for extensive biophysical characterization. It involves coexpressing in the same plasmid (YEp213) the nonfunctional gene with a functional gene copy for complementation in a selective medium. The functional gene encodes a product with an engineered metal-chelating dihistidine site (His-39 and Leu-58-->His) that enables efficient separation of the two isoforms by immobilized metal-affinity chromatography. The purified Met-80-->Ala protein possesses a binding site for dioxygen and other exogenous ligands. Absorption spectra of several derivatives of this mutant show striking similarities to those of corresponding derivatives of horseradish peroxidase, myoglobin, and cytochrome P450. The use of a dual-gene vector for cytochrome c expression together with metal-affinity separation opens the way for the engineering of variants with dramatically altered structural and catalytic properties.
Graduate Work
5 | Yi Lu, James A. Roe, Christopher J. Bender, Jack Peisach, Lucia Banci, Ivano Bertini, Edith B. Gralla, Joan Selverstone Valentine
"New Type 2 Copper-Cysteinate Proteins. Copper Site Histidine-to-Cysteine Mutants of Yeast Copper−Zinc Superoxide Dismutase"
Inorganic Chemistry 35, 1692-1700 (1996) [PDF] [archived PDF] [doi]

Two new site-directed mutant copperzinc superoxide dismutase proteins, i.e., His46Cys and His120Cys, have been prepared. They are characterized by their yellow color and their type 2 EPR and visible absorption spectra. An interesting difference between them is that the imidazolate bridge that is characteristic of the wild-type protein remains intact in the case of the H46C mutant but is not present in the case of the H120C mutant.
4 | Yi Lu, Louis B. LaCroix, Michael D. Lowery, Edward I. Solomon, Christopher J. Bender, Jack Peisach, James A. Roe, Edith B. Gralla, Joan Selverstone Valentine
"Construction of a blue copper site at the native zinc site of yeast copper-zinc superoxide dismutase"
Journal of the American Chemical Society 115, 5907-5918 (1993) [PDF] [archived PDF] [doi]
3 | Yi Lu, James A. Roe, Edith G. Gralla, Joan Selverstone Valentine
"Metalloprotein Ligand Redesign: Characterization of Copper-Cysteinate Proteins Derived from Yeast Copper-Zinc Superoxide Dismutase"
in "Bioinorganic Chemistry of Copper", Kenneth D. Karlin, Zoltan Tyeklar, eds.; Chapman & Hall: New York; pp. 64-77 (1993)
2 | Jane Han, Thomas M. Loehr, Yi Lu, Joan Selverstone Valentine, Bruce A. Averill, Joann Sanders-Loehr
"Resonance Raman excitation profiles indicate multiple Cys -> Cu charge transfer transitions in type 1 copper proteins"
Journal of the American Chemical Society 115, 4256-4263 (1993) [PDF] [archived PDF] [doi]
1 | Yi Lu, Edith B. Gralla, James A. Roe, Joan Selverstone Valentine
"The redesign of a type 2 into a type 1 copper protein: construction and characterization of yeast copper, zinc superoxide dismutase mutants"
Journal of the American Chemical Society 114, 3560-3562 (1992) [PDF] [archived PDF] [doi]