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 two prokaryotic repressor proteins: the P22 Arc protein and the N-terminal domain of lambda repressor. These proteins provide excellent model systems because their three-dimensional structures are known, the systems are easily manipulated genetically, and biochemical and biophysical studies of the mutant proteins are straightforward. Single or multiple mutations that alter hydrophobic packing interactions or salt-bridge networks in both proteins will be constructed. The effects of these mutations on thermodynamic stability will be investigated by reversible denaturation experiments; changes in the kinetics of folding and unfolding for Arc variants will be studied by stop-flow methods. The conformational properties of the mutants will be probed by DNA binding activity, the binding of conformation specific monoclonal antibodies, and in some cases by NMR and/or x-ray crystallography. The cooperativity of stabilizing interactions will be investigated by construction of thermodynamic cycles for sets of singly and multiply mutant proteins. Some studies will employ saturation mutagenesis methods to allow statistical analysis of interactive sequence information. Since stability results from the difference between the free energies of the folded and unfolded protein conformations, the conformational properties of Arc variants denatured by dilution will also be determined using circular dichroism, fluorescence, and gel filtration chromatography. These studies will provide an important database for testing and refining our understanding of the sequence determinants of protein structure and stability and for evaluating algorithms that seek to predict the effects of sequence changes on protein structure and function.