| Incorporation of non-natural amino acids into proteins in vivo can provide biological materials with new chemical functions and improved physical properties. Examples include new posttranslational modification chemistry by introducing azide and ketone moieties into recombinant proteins, and novel strategies for engineering hyperstable proteins by incorporating fluorinated side chains. Implementing such methods requires manipulation of protein biosynthesis to specifically alter the genetic code. The rules of the genetic code are established by the aminoacylation reaction, where the aminoacyl-tRNA synthetases (aaRS) catalyze the attachment of the amino acids to their cognate tRNAs. Thus manipulation of cellular aminoacylation reactions could potentially expand the available set of amino acid building blocks for protein engineering and biomaterials engineering.; By simple depletion of the cellular pool of isoleucine and utilization of isoleucine auxotrophic hosts, we were able to force the endogenous isoleucyl-tRNA synthetase to join 5,5,5-trifluoroisoleucine (5TFI) to tRNAIle and assign it to isoleucine codons in vivo. Murine interleukin 2 containing 5TFI retains its biological activity. We showed that engineering of bacterial expression hosts can allow a single RNA message to be read in different ways, depending on the relative rates of competing aminoacylation reactions. Specifically, we showed that the 2S,3R-form of 4,4,4-trifluorovaline can be assigned either to isoleucine or to valine codons, depending on whether the bacterial host overexpresses the isoleucyl- or the valyl-tRNA synthetase. When an amino acid analog of interest is not recognized by the corresponding wild-type aaRS, we can either identify the appropriate modification of the amino acid as a promising ligand or design new synthetase activity. We describe an attempt to develop a virtual ligand screening method to find non-natural amino acids that can serve as ligands for the phenylalanyl-tRNA synthetase and our computational results correlate well with experimental results in vitro and in vivo. We also present a computational method for identifying the sites of mutations to relax the substrate specificity of the E. coli phenylalanyl-tRNA synthetase (ePheRS). One designed variant of ePheRS allows the efficient in vivo incorporation of aryl ketone functionality into proteins in vivo. (Abstract shortened by UMI.)... |
