The goal of this work is to measure the energetic contributions to the stabilization of protein structures made by specific interactions within these structures. This knowledge will be of considerable utility in the design of medically and industrially important synthetic hormones, vaccines, regulatory protein, and enzymes. This project will use the gene V (DNA-binding) protein of bacteriophage f1 as a model system to measure the effects of amino-acid substitutions on protein stability and structure. First, we plan to obtain an accurate structure of the gene V protein by x-ray diffraction analysis. An accurate model will be essential for interpretation of our previous experiments and as a starting model for determining the structures of mutant gene V proteins. New phasing information will be obtained from several techniques including collection of x-ray diffraction data from selenomethionine-containing gene V protein crystals at several wavelengths. A model obtained from these procedures will be refined against 1.8angstroms native data. Next we plan to use this new structure of the gene V protein to interpret our previous results. We propose to use this structure to formulate hypotheses as to why some gene V temperature-sensitive mutants have altered stabilities, to interpret effects of apolar-to-apolar substitutions at buried sites, and to interpret effects of valine to threonine substitutions at interior sites. Third, we will determine the crystal structures of strongly stabilized and destabilized mutants. We previously used a genetic approach to identify interactions that are particularly strong in the gene V protein. We propose to crystallize 11 mutants we have already purified that exhibit very different stabilities than the wild-type. Using the structures of the mutant proteins, we anticipate being able to suggest the structural basis of their changes in stability. Finally, we will construct mutants to measure energetic effects due to specific interations in the gene V protein. We propose to measure the contribution to changes in stability from specific interactions by constructing additional mutants which differ from each in the property of interest, such as electrostatic interactions or hydrogen bonding, and determining their stabilities and structures, as well as by examining control mutants that do not differ in the property of interest. By comparing the contributions of a particular interaction in different contexts within the protein, the geometric or solvent exposure requirements for making the interaction can be evaluated.
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