Theoretical calculations were carried out to provide a framework for understanding the free radical oxidation of unsaturated lipids. The carbon[bond]hydrogen bond dissociation enthalpies (BDEs) of organic model compounds and oxidizable lipids (R[bond]H) and the carbon[bond]oxygen bond dissociation enthalpies of peroxyl radical intermediates (R[bond]OO*) have been calculated. The carbon[bond]hydrogen BDEs correlate with the rate constant for propagation of free radical autoxidation, and the carbon[bond]oxygen BDEs of peroxyl radicals correlate with rate constants for beta-fragmentation of these intermediates. Oxygen addition to intermediate carbon radicals apparently occurs preferentially at centers having the highest spin density. The calculated spin distribution therefore provides guidance about the partitioning of oxygen to delocalized carbon radicals. Where the C[bond]H BDEs are a function of the extent of conjugation in the parent lipid and the stability of the carbon radical derived therefrom, C[bond]OO* BDEs are also affected by hyperconjugation. This gives way to different rates of beta-fragmentation of peroxyl radicals formed from oxygen addition at different sites along the same delocalized radical. We have also studied by both theory and experiment the propensity for benzylic radicals to undergo oxygen addition at their ortho and para carbons which, combined, possess an equivalent unpaired electron spin density as the benzylic position itself. We find that the intermediate peroxyl radicals in these cases have negative C[bond]OO* BDEs and, thus, have rate constants for beta-fragmentation that exceed the diffusion-controlled limit for the reaction of a carbon-centered radical with oxygen.

We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in V H CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded ''synthetic'' genetic code can confer a selective advantage in the directed evolution of proteins with specific properties. directed evolution ͉ phage display ͉ unnatural amino acids W ith few exceptions, the genetic codes of all known organisms specify only the 20 canonical amino acids for protein synthesis. Yet it is quite possible that the ability to encode additional amino acids and their corresponding chemical functionalities would be evolutionarily advantageous, especially since nature's choice of 20 could have been arbitrarily fixed at the point of transition between communal and Darwinian evolution paradigms and subsequently sustained by the code's inertia (1). Furthermore, in the limited scope of laboratory-directed evolution, which concerns only one or few specific functions over a short time rather than general organismal fitness over thousands of years, one can easily envision a selective advantage conferred by additional amino acids. Recent developments in our laboratory allow us to explore this possibility. Specifically, orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs capable of incorporating various unnatural amino acids into proteins in response to unique nonsense and frameshift codons have been added to the translational machinery of Escherichia coli (2). These E. coli (X-E. coli) can now be used for evolution of protein function wherein 21 building blocks, rather than the common 20, are available.Several unnatural amino acids were initially chosen, on the basis of their unique chemistries, for use in our system. For example, X-E. coli genetically encoding the bidentate metalchelating amino acid bipyridyl-alanine (3) are well-suited for the evolution of redox and hydrolytic catalysts, as metal ion binding would not require preorganized primary and secondary ligand shells. Similarly, X-E. coli encoding the reactive 4-boronophenylalanine (4) are well-suited for evolution of receptors specific for glycoproteins or serine protease inhibitors, because the boronate group can form covalent complexes with diols or reactive serine residues. In addition, X-E. coli genetically encoding otherwise posttranslationally modified amino acids, such as sulfotyrosine (5), can be used for evolution of properties that exploit the unique chemical characterist...

Genetically encoded unnatural amino acids could facilitate the design of proteins and enzymes of novel function, but correctly specifying sites of incorporation, and the identities and orientations of surrounding residues represents a formidable challenge. Computational design methods have been used to identify optimal locations for functional sites in proteins and design the surrounding residues, but have not incorporated unnatural amino acids in this process. We extended the Rosetta design methodology to design metalloproteins in which the amino acid (2,2’-bipyridin-5yl)alanine (Bpy-Ala) is a primary ligand of a bound metal ion. Following initial results that indicated the importance of buttressing the Bpy-Ala amino acid, we designed a buried metal binding site with octahedral coordination geometry consisting of Bpy-Ala, two protein based metal ligands, and two metal bound water molecules. Experimental characterization revealed a Bpy-Ala mediated metalloprotein with the ability to bind divalent cations including Co2+, Zn2+, Fe2+, and Ni2+, with a Kd for Zn2+ of ~40 pM. X-ray crystallographic analysis of the designed protein shows only slight deviation from the computationally designed model.

The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a kcat/KM of 568 M–1 s–1, representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.

We recently developed a phage-based system for the evolution of proteins in bacteria with expanded amino acid genetic codes. Here, we demonstrate that the unnatural amino acid p-boronophenylalanine (BF) confers a selective advantage in the evolution of glycan binding proteins. We show that an unbiased library of naïve antibodies with NNK-randomized VH CDR3 loops converges upon mutants containing BF when placed under selection for binding to a model acyclic amino sugar. This work represents a first step in the evolution of carbohydrate binding proteins that use a reactive unnatural amino acid “warhead” and demonstrates that a “synthetic” genetic code can confer a selective advantage by increasing the number of functional groups available to evolution.