Physico-Chemical Studies of Proteins and Nucleic Acids

Koeppe, Roger

Abstract

Research involving mechanistic protein chemistry will be focused in two areas: the chemical control of ion transport through gramicidin channels, and identification of the catalytic site of Trp-tRNA synthetase.
The specific aims of the two projects are to better understand the molecular basis of rapid, selective ion transport across biological membranes, and to describe the chemical basis for specific nucleic acid (RNA) recognition by a particular enzyme (Trp-tRNA synthetase). The structural, transport and equilibrium ion binding properties of chemically modified analogues of gramicidin will be analyzed by NMR, single-channel, diffraction and sequencing techniques. An exciting new naturally-occurring, channel-forming gramicidin from Bacillus brevis which has recently been discovered in our laboratory will be further characterized. This new product will lend insight into the mechanism of biosynthesis of gramicidins in the bacterium. Other gramicidin analogues will be synthesized for comparisons of the relative influence of ion-dipole interactions as opposed to electron inductive effects on the transport process. The sites of covalent inhibition of Trp-tRNA synthetase by mesitoyl-AMP and by analogues of tryptophan will be mapped by protease digestion and HPLC techniques. To produce more of the enzyme, and to prepare for subsequent DNA modifications to specifically alter the amino acid sequence of the enzyme, we will place the gene of interest under control of the Lac operator.