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...
[a] Photoaffinity labels and crosslinkers have been used to map biomolecular interactions, as well as to identify the biological targets of small molecules. [1][2][3][4] Benzophenones are among the most useful photocrosslinking agents and preferentially insert into CÀH bonds upon excitation with UV light. Aryl azides and [3-(trifluoromethyl)-3H-diazin-3yl]phenones generate reactive nitrenes and carbenes, respectively, that undergo relatively nonspecific insertion and addition reactions. [5,6] To facilitate the selective incorporation of photocrosslinking agents into proteins in living cells, we recently genetically encoded para-benzoyl-l-phenylalanine (pBpa) and para-azido-l-phenylalanine (pAzpa) in response to the amber nonsense codon in E. coli, yeast and mammalian cells. [7][8][9][10][11][12] These photocrosslinkers were site-specifically incorporated into proteins by means of heterologous amber suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pairs that recognize the unnatural amino acid, but do not cross-react with endogenous host cell tRNAs, aaRSs or amino acids. To expand the photoaffinity label repertoire, we now report that 4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]-l-phenylalanine (TfmdPhe; Figure 1 A) can also be genetically encoded with excellent efficiency and fidelity in bacteria.TfmdPhe has useful photochemical properties and is stable under physiological conditions. Upon excitation by~350 nm light, TfmdPhe undergoes fragmentation to N 2 and a reactive carbene, which readily inserts into CÀH or OÀH bonds. [13][14][15] In contrast, pAzpa requires relatively short wavelength UV excitation and can rearrange to less reactive secondary products prior to crosslinking. TfmdPhe is also somewhat smaller than pBpa, which can facilitate its incorporation into proteins at sites that are involved in biomolecular interactions. TfmdPhe has been incorporated into proteins previously by a chemically misacylated amber suppressor tRNA in a cell-free protein expression system.[16] However, this method severely limits the protein yield, and is not amenable to studies directly in living cells.TfmdPhe was synthesized by using a previously reported method.[17] To selectively incorporate TfmdPhe into proteins in E. coli, an orthogonal amber suppressor tRNA/aaRS was generated from a Methanococcus jannaschii amber suppressor tRNA (MjtRNA CUA )/para-bromo-l-phenylalanyl-tRNA synthetase (BrPheRS) pair by using a previously reported, directed evolution strategy. BrPheRS was used as a template for mutagenesis because para-bromo-l-phenylalanine (BrPhe) is structurally similar to TfmdPhe, and BrPheRS has polypeptide backbone A C H T U N G T R E N N U N G rearrangements that enlarge the substrate-binding pocket. Based on the structure of the BrPheRS-BrPhe complex, [18] a library of aaRS active-site mutants was generated, in which residues L32, L65, H70, Q109, H160 and Y161 of BrPheRS were randomized by overlap extension polymerase chain reaction with synthetic oligonucleotide primers; the intended mutations were encoded by NNK (...
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.
A wide array of amino acids with novel chemical and biological properties have been genetically encoded in both prokaryotic and eukaryotic organisms, [1][2][3] which include the efficient photo-cross-linker para-benzoyl-l-phenylalanine (pBpa, Figure 1). Orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs that selectively recognize pBpa have been evolved from both Methanococcus jannaschii (Mj) and Escherichia coli (Ec) tyrosyl-tRNA synthetases (TyrRS) in bacteria and yeast, respectively. [4,5] To understand the structural basis for selective recognition of the large benzophenone side chain by these mutant enzymes, we now report the X-ray crystal structure of the mutant MjTyrRS-pBpa complex. A model of the corresponding mutant EcTyrRS-pBpa complex was also generated and shares many features with the M. jannaschii structure. In contrast to previous structural studies of evolved aminoacyl-tRNA synthetases, these mutant enzymes bind the relatively large side chain of pBpa in a deep hydrophobic cavity with relatively little change in the polypeptide backbone.To evolve a pBpa-specific Mj aminoacyl-tRNA synthetase (MjpBpaRS) in E. coli, a pool of MjTyrRS variants was generated with random mutations at five active-site amino acid residues (Y32, E107, D158, I159, and L162). Alternating rounds of positive and negative selection were then used to identify mutant aaRSs that aminoacylate an amber suppressor tRNA CUA with pBpa, but not with endogenous host amino acids. [6,7] After five rounds of selection, six MjpBpaRSs were identified that showed sequence convergence. In most of these clones, Y32 was mutated to alanine or glycine; D158 was mutated to threonine; I159 was mutated to serine; and L162 was conserved; Y32G/D158T/I159S emerged as the consensus set of mutations. A similar method was used to evolve an EcTyrRS that selectively charges its cognate suppressor tRNA CUA with pBpa in yeast (EcpBpaRS). Positive and negative selections of a library that contains random mutations at five active-site residues (Y37, N126, D182, F183, and L186) afforded two clones selective for pBpa. In both clones, Y37 was mutated to glycine, N126 was conserved, D182 was mutated to glycine, F183 was either conserved or mutated into tyrosine, and L186 was mutated to alanine or methionine. Given the structural homology between archael and bacterial tyrosyl-tRNA synthetases, and the fact that the mutations to each are quite similar, it is likely that MjpBpaRSs and EcpBpaRSs bind the side chain of pBpa in a similar fashion. To address this question, the X-ray crystal structure of the MjpBpaRS (Y32G/D158T/I159S)-pBpa complex was determined, and the corresponding structure of the EcpBpaRS (Y37G/D182G/L186A)-pBpa complex was modeled.MjpBpaRS was crystallized in the presence of 1 mm pBpa by the hanging-drop vapor-diffusion method. The protein crystals belong to the space group P4 3 2 1 2 and contain one molecule per asymmetric unit (Table 1). There is one molecule of pBpa per protein bound at the active site as evidenced by the strong Fo-Fc electron d...
A wide array of amino acids with novel chemical and biological properties have been genetically encoded in both prokaryotic and eukaryotic organisms, [1][2][3] which include the efficient photo-cross-linker para-benzoyl-l-phenylalanine (pBpa, Figure 1). Orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs that selectively recognize pBpa have been evolved from both Methanococcus jannaschii (Mj) and Escherichia coli (Ec) tyrosyl-tRNA synthetases (TyrRS) in bacteria and yeast, respectively. [4,5] To understand the structural basis for selective recognition of the large benzophenone side chain by these mutant enzymes, we now report the X-ray crystal structure of the mutant MjTyrRS-pBpa complex. A model of the corresponding mutant EcTyrRS-pBpa complex was also generated and shares many features with the M. jannaschii structure. In contrast to previous structural studies of evolved aminoacyl-tRNA synthetases, these mutant enzymes bind the relatively large side chain of pBpa in a deep hydrophobic cavity with relatively little change in the polypeptide backbone.To evolve a pBpa-specific Mj aminoacyl-tRNA synthetase (MjpBpaRS) in E. coli, a pool of MjTyrRS variants was generated with random mutations at five active-site amino acid residues (Y32, E107, D158, I159, and L162). Alternating rounds of positive and negative selection were then used to identify mutant aaRSs that aminoacylate an amber suppressor tRNA CUA with pBpa, but not with endogenous host amino acids. [6,7] After five rounds of selection, six MjpBpaRSs were identified that showed sequence convergence. In most of these clones, Y32 was mutated to alanine or glycine; D158 was mutated to threonine; I159 was mutated to serine; and L162 was conserved; Y32G/D158T/I159S emerged as the consensus set of mutations. A similar method was used to evolve an EcTyrRS that selectively charges its cognate suppressor tRNA CUA with pBpa in yeast (EcpBpaRS). Positive and negative selections of a library that contains random mutations at five active-site residues (Y37, N126, D182, F183, and L186) afforded two clones selective for pBpa. In both clones, Y37 was mutated to glycine, N126 was conserved, D182 was mutated to glycine, F183 was either conserved or mutated into tyrosine, and L186 was mutated to alanine or methionine. Given the structural homology between archael and bacterial tyrosyl-tRNA synthetases, and the fact that the mutations to each are quite similar, it is likely that MjpBpaRSs and EcpBpaRSs bind the side chain of pBpa in a similar fashion. To address this question, the X-ray crystal structure of the MjpBpaRS (Y32G/D158T/I159S)-pBpa complex was determined, and the corresponding structure of the EcpBpaRS (Y37G/D182G/L186A)-pBpa complex was modeled.MjpBpaRS was crystallized in the presence of 1 mm pBpa by the hanging-drop vapor-diffusion method. The protein crystals belong to the space group P4 3 2 1 2 and contain one molecule per asymmetric unit (Table 1). There is one molecule of pBpa per protein bound at the active site as evidenced by the strong Fo-Fc electron d...
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