BIOPHYSICAL STUDIES OF FOLDING MUTANTS OF STAPH NUCLEASE

本申请所述研究的长期目标是

The long term objective of the research described in this application is the development of a quantitative model to describe how the 149 amino acids of staphylococcal nuclease determine the structure of the native state, its stability, and its folding pathway. To achieve this objective, extensive use will be made of genetic engineering methods to systematically alter this small protein's amino acid sequence in a variety of ways. 1) Each amino acid residue will be mutated to both alanine and glycine to remove the wild-type side chain. 2) Single alanine and glycine residues will be inserted between a number of pairs of wild-type residues to alter the spacing between chain segments. 3) Many of the 20 amino acids will be substituted at several select positions to examine the effect of different side chains. 4) Randomly induced mutations that lower the stability of nuclease to reversible denaturation will be recovered using a simple plate assay. To quantitate the effects of these sequence modifications on structure and folding, a variety of biophysical methods will be used in the characterization of highly purified mutant proteins. As a first step, the free energy change deltaG on denaturation and the rate of change of delta G with respect to denaturant concentration will be determined by monitoring the equilibrium unfolding reaction via intrinsic fluorescence and circular dichroism. Correlations will then be sought between these two values and a number of parameters that describe a residue's local environment in the native state. For unusual mutants such as stable insertions, the structural consequences will be identified by x-ray crystallographic methods in collaboration with other laboratories. Mutants that appear to alter the residual structure of the denatured state will be recombined into large fragments of nuclease ( which serve as models of the denatured state) and their residual structure quantitated by circular dichroism and gel filtration. To examine mutant effects on the denatured state of full length protein, the techniques of fluorescence energy transfer will be used. A detailed study of a presumptive intermediated state of folding dominated by hydrophobic interactions will be continued, including structural studies by high resolution proton NMR. Kinetic analysis of the rates of folding and unfolding will be initiated. And the patterns of hydrophobic contacts in a number of small proteins, including staph nuclease mutants, will be characterized with the mathematical tools of Graph Theory.