Most physiological processes rely on the function of proteins. In order to elucidate the precise biochemical mechanisms of protein function, researchers are increasingly turning to the tool set of unnatural amino acids that contain small structural or electronic changes which are very similar to Nature's set of common amino acids. These subtle differences, site-specifically incorporated into proteins, allow for the precise probing of protein structure and function. Within this project, new methodologies for the genetic encoding of these 'near-natural' amino acids will be developed and applied to the investigation of enzymatic mechanism.

In order to elucidate the precise biochemical mechanisms of protein function, unnatural amino acids that contain small structural or electronic changes, e.g., in pKa, polarity, redox potential, H-bonding ability, nucleophilicity, isotope composition, etc, are versatile molecular probes. Although these 'near-natural' amino acids are extremely useful to investigate protein function when located near or in an active site, they are difficult to apply to the engineering of a biological system for the following reasons: 1) The chemical synthesis of proteins containing near-natural amino acids is laborious, expensive, and can routinely only be applied to small proteins. 2) The semi-synthesis of proteins can be technically challenging. 3) The use of auxotrophic bacterial or yeast strains in vivo leads to global, non-specific incorporation of near-natural amino acids into proteins preventing precise studies. 4) Near-natural amino acids often display toxicity due to global incorporation into the proteome. Within this project, a robust and general methodology for protein engineering through site-specific near-natural amino acid mutagenesis will be developed by temporarily disguising these amino acids as completely unnatural amino acids. This will be achieved by introducing a transient structural change into near-natural amino acids, thus 'hiding' them from the cellular machinery responsible for endogenous protein biosynthesis, but simultaneously providing a handle for the engineering of an orthogonal biosynthetic pathway. Specifically, introduction of protecting groups (caging groups) on near-natural amino acids converts them temporarily into completely unnatural amino acids, effectively solving the issue of non-specific incorporation into proteins in vivo. After site-specific incorporation into the protein of interest, the caging group is removed through an external trigger providing the original near-natural amino acid and thus near-natural protein. For the engineering of an orthogonal protein biosynthetic pathway, new tRNA synthetases for the in vivo incorporation of near-natural amino acids into proteins in pro- and eukaryotic cells are generated. The developed methodology will be applied to the precise investigation of enzyme active sites with atomic resolution.

This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Division of Materials Research through BioMaPS funds.

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University of Pittsburgh
United States
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