The goal of this work is to understand the mechanisms of folding of proteins and the interactions determining their stabilities. This knowledge will be of great use in the design of medically important synthetic hormones and vaccines and in the design of scientifically and commercially important regulatory proteins, enzymes and structure proteins. This project will use the gene V (DNA-binding) protein of bacteriophage f as a model system to examine the effects of amino-acid substitutions on protein stability and folding, and to identify the role of intermediate structures in protein folding. Mutant proteins will be obtained by directed mutagensis and by direct genetic selection for temperature-sensitive folding and stability. These mutant proteins will be purified and characterized by rapid size-exclusion chromatography, rates of proteolytic cleavage at various temperatures, and calomimetry and two-dimensional NMR ( in collaboration). Directed mutagnesis will be used to examine three properties of the amino acids in the gene V protein for their importance in the stability of the protein. These are the sizes of buried apolarside chains, the conformational preferences of the amino acids, and the charges on the amino-acid side chains. In each case, one or a group of amino-acid substitutions will be made which preferentially alter the property to be evaluated, and the resulting changes in the stability of the protein and its kinetics of folding will be measured. A more detailed analysis of the causes of these changes in stability will be carried out using a combination of potential energy calculations and molecular graphics. Genetic selections for and against the activity of the gene V protein recently demonstrated here will be used to develop the first direct genetic selections for temperature-sensitive stability and folding of a protein. Using these genetic selections, mutant gene V proteins which are temperature-sensitive for stability, and second-site mutations which suppress this phenotype will be isolated and characterized. The properties of these mutant proteins will be used to test the conclusions reached from the directed mutagensis experiments and to suggest other types of interactions which are important in protein stability. Finally, mutant gene V proteins which are temperature-sensitive for folding but not for stability will be isolated and used to identify interactions which are important only in intermediate stages of folding of the protein.
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