Unnatural Amino Acids and Non-Native Cofactors
Figure 1
A closer look at native metalloproteins reveals that they use only a small sub-set of the ligands and metal-containing prosthetic groups synthesized by inorganic chemists. For example, there are only 20 natural amino acids, less than half of which are capable of coordinating to metal ions, and the number of metal-containing cofactors is also limited. Unnatural amino acids or non-native metal-containing cofactors greatly expand the range of chemical moieties and functionality of a single protein without grossly changing the overall structure of the site it replaces. The precise roles of key residues important to protein structure and function can be fine-tuned beyond what is found in nature. Using the same technique, we incorporated several derivatives of Tyr in place of Tyr33 in our F33Y-CuBMb model and showed the important role of the pKa and redox potential of the active site Tyr residue (1, 2).

We became the first lab to introduce unnatural amino acids into metalloproteins using a method called expressed protein ligation (EPL) (3-5). We have also contributed to the field by expanding the EPL method by, for example, developing protocols for using selenocysteine instead of cysteine as the coupling amino acid (3, 6). Isosteric substitutions using UAAs enabled us to resolve long-standing issues such as the role of conserved methionine and cysteine in blue copper azurin (3, 8-11). Our analysis revealed hydrophobicity as the dominant factor in tuning the reduction potentials of blue copper centers by axial ligands (8) and the role of the axial ligand in tuning site geometry (10) (Figure 1). Recently, we expanded our ability to incorporate UAAs by using an orthologous tRNA-tRNA synthetase pair. Using this method an UAA was incorporated into CuBMb (our HCO model) that mimics the native HCO His-Tyr crosslink. Introduction of this crosslink increased the rate and efficiency of the reaction significantly (12).

Figure 2
One of the greatest challenges faced by today's synthetic chemists is the search for ways to carry out chemical syntheses both asymmetrically and in an environmentally benign way. We have developed a novel dual-anchor approach to introduce non-native cofactors such as Mn(Salen) (13) (see Figure 2) or metallocenes (14) into proteins to produce novel biocatalysts with unprecedented structural and functional properties. The combination of the high catalytic reactivity of metal complexes with the rigid and chiral environment of a protein allows us to design and engineer a new generation of catalysts with novel reactivity and selectivity. In addition, through systematic variation of the protein environment around both the metal- and substrate-binding sites, we can tailor reactivity and regio-, stereo-, and enantio-selectivity with high precision (4, 15-18).
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