Recently, tRNA aminoacyl-tRNA synthetase pairs have been evolved that allow one to genetically encode a large array of unnatural amino acids in both prokaryotic and eukaryotic organisms. We have determined the crystal structures of two substrate-bound Methanococcus jannaschii tyrosyl aminoacyl-tRNA synthetases that charge the unnatural amino acids p-bromophenylalanine and 3-(2-naphthyl)alanine (NpAla). A comparison of these structures with the substratebound WT synthetase, as well as a mutant synthetase that charges p-acetylphenylalanine, shows that altered specificity is due to both side-chain and backbone rearrangements within the active site that modify hydrogen bonds and packing interactions with substrate, as well as disrupt the ␣8-helix, which spans the WT active site. The high degree of structural plasticity that is observed in these aminoacyltRNA synthetases is rarely found in other mutant enzymes with altered specificities and provides an explanation for the surprising adaptability of the genetic code to novel amino acids.x-ray crystal structure ͉ unnatural amino acids ͉ expanded genetic code ͉ molecular evolution W ith the rare exceptions of selenocysteine (1) and pyrrolysine (2, 3), the common 20 amino acids are conserved across all known organisms. However, there does not appear to be an inherent limit to the size or chemical nature of the genetic code, since it has been shown that additional amino acids can be genetically encoded in both prokaryotic and eukaryotic organisms in response to nonsense or frameshift codons (4, 5). This requires a unique codon-suppressor tRNA pair and the corresponding aminoacyl-tRNA synthetase, which do not crossreact with the amino acids, tRNAs, or synthetases of the host organism (4, 5). The specificity of the aminoacyl tRNA synthetase is then altered by generating large libraries of active-site mutants and passing them through positive and negative selections to identify synthetases that selectively acylate the cognate tRNA with the unnatural amino acid but not any of the common amino acids. This approach has been used to add Ͼ30 unnatural amino acids to the genetic codes of bacteria, yeast, and mammalian cells with high fidelity and efficiencies.In Escherichia coli, a tyrosyl aminoacyl-tRNA synthetase (TyrRS) tRNA CUA Tyr pair from the archea Methanococcus jannaschii (Mj) was used as an orthogonal tRNA synthetase pair (6). Mutant synthetases have been evolved that selectively aminoacylate their cognate suppressor tRNAs with glycosylated (7), photoreactive (8), and metal-binding amino acids, as well as amino acids with unique functional groups (9-11) . To establish the molecular basis for the surprising adaptability of this synthetase, we solved (12) the structure of a mutant Mj TyrRS that is selective for p-acetylphenylalanine (p-AcPhe). The x-ray crystal structure revealed significant structural changes within the enzyme active site that result from the mutations Y32L, D158G, I159C, and L162R. The Y32L and D158G mutations remove two hydrogen bonds (H-bonds) with the...

It has been recently shown that orthogonal tRNA/aminoacyl-tRNA synthetase pairs can be evolved to allow genetic incorporation of unnatural amino acids into proteins in both prokaryotes and eukaryotes. Here we describe the crystal structure of an evolved aminoacyl-tRNA synthetase that charges the unnatural amino acid p-acetylphenylalanine. Molecular recognition is due to altered hydrogen bonding and packing interactions with bound substrate that result from changes in both side-chain and backbone conformation.

A protein evolution strategy is described by which double-stranded DNA fragments encoding defined E. coli protein secondary structural elements (α-helices, β-strands and loops) are assembled semirandomly into sequences comprised of as many as 800 amino acid residues. A library of novel polypeptides generated from this system was inserted into an EGFP fusion vector and members screened by FACS to identify those polypeptides that fold into soluble, stable structures in vivo that comprised a subset of shorter sequences (∼60 to 100 residues) from the semi-random sequence library. Approximately 10 8 clones were screened by FACS, a set of 1149 high fluorescence colonies were characterized by dPCR and four soluble clones with varying amounts of secondary structure were identified. One of these is highly homologous to a domain of aspartate racemase from a marine bacterium (Polaromonas sp.) but is not homologous to any E. coli protein sequence. Several other selected polypeptides have no global sequence homology to any known protein, but show significant α-helical content but limited dispersion in 1D NMR spectra, pH sensitive ANS binding and reversible folding into soluble structures. These results demonstrate that this strategy can generate novel polypeptide sequences containing secondary structure.Despite the large sequence diversity present in the proteomes of known organisms, most functional proteins characterized to date assume one of relatively few distinct folds [1][2][3][4][5] , suggesting that nature uses a limited number of stable, soluble folds relative to what is theoretically possible in protein sequence space. These folds likely represent the divergent products of a limited set of ancient protein folds. However, protein sequences that fold into stable, unique topologies but are not encoded by any sequenced genome may also exist. It may be possible to identify other stable folds that simply have not yet been sampled in the course of evolution by means of an artificial selection and molecular evolution process. Methods such as DNA shuffling 6 which mimic the combinatorial diversity mechanism of the immune system are among the most efficient methods to modify or enhance protein activity. Unfortunately, the structural diversity generated in a library of shuffled homologues is relatively small 7-11 , *To whom correspondence should be addressed. Telephone: 858-784-9273 (P.G.S.), 858-812-1551 (S.A.L.), or 858-812-1633 (B.H.G.). Fax: 858-784-9440 (P.G.S.), 858-812-1920 (S.A.L.), or 858-812-1746 (B.H.G.). E-mail: schultz@scripps.edu; E-mail: slesley@gnf.org; E-mail: bgeierstanger@gnf.org. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript although newer methods for random fragment assembly that overcome sequence homology requirements may lead to libraries with increased structural diversity 12,13 . Alternatively, natural or in vitro combinatorial assembly of distinct protein subunits (e.g., subdomains, exons, etc.) can create significant structural and functional diversity [14][15][16...