(6l) Proteins of Novel Composition

The properties of proteins are strikingly susceptible to engineering via rational and combinatorial strategies. In chemical terms, however, the engineering strategies employed to date are remarkably conservative, in that they employ a common set of twenty ?canonical? amino acid constituents. Recent work from several laboratories has shown that a substantially expanded repertoire of non-canonical amino acids can be incorporated into recombinant proteins, either by amino acid replacement or by nonsense or frameshift suppression. This work raises intriguing questions about the prospects for creating novel proteins by recruitment of new amino acid constituents. In particular, global replacement of one of the canonical amino acids by a non-canonical analog would be expected to cause marked changes in protein structure, dynamics, and function. The subject of my thesis research (conducted in the laboratory of David Tirrell) has focused on three specific topics in the area of protein engineering via global incorporation of noncanonical amino acids: 1) Green fluorescent protein was engineered to tolerate replacement of Leu by 5,5,5-trifluoroleucine (Tfl) by using a combination of global incorporation of Tfl and directed evolution; 2) A high-throughput method of screening aminoacyl-tRNA synthetase libraries has been developed, and the strategy was demonstrated by identifying an Escherichia coli methionyl-tRNA synthetase variant that activates 6,6,6-trifluoronorleucine; 3) The effects of Tfl on hydration dynamics in a coiled-coil protein (A1 protein) are currently being examined in collaboration with the Zewail group at the California Institute of Technology.