The long term objective of this research is to understand the relationship between the amino acid sequence of a protein and the ability of the protein to fold into a stable, three dimensional structure. This is important because an understanding of protein stability and folding underlies any detailed understanding of most biological reactions. Moreover, much of the promise of biotechnology for improving human health depends on our eventual ability to manipulate the function of a protein by altering its sequence. The studies described here will probe the determinants of protein folding and stability for three repressor proteins: the lambda Cro protein, the N-terminal domain of lambda repressor, and the P22 Arc protein. These proteins provide excellent model systems because their structures are known or will be known soon, the systems are easily manipulated genetically, and biochemical and biophysical studies of the mutant proteins are straightforward. Our studies will employ classical or modern mutagenesis methods to create populations of singly or multiply mutant proteins, from which we will screen or select for proteins with interesting structural phenotypes. For example, techniques of reversion analysis and immuno-screening will be applied to obtain proteins with enhanced stabilities, and combinatorial cassette mutagenesis and genetic selection will be used to identify libraries of sequences that can form beta-turns, mediate symmetric helix/helix packing, or allow highly efficient nucleation of alpha- helix formation. Equilibrium constants for denaturation and dimerization of the mutant proteins will be determined, and in some cases peptide synthesis or site-directed mutagenesis will be used to test specific hypothesis concerning the molecular mechanisms of the observed changes in structure or stability. The structures of interesting mutant proteins will be investigated by collaborative crystallographic or nuclear magnetic resonance experiments.
Showing the most recent 10 out of 98 publications
