The long term objective of the research described in this application continues to be the development of a quantitative model that describes how the 149 amino acids of staphylococcal nuclease determine the structure of its native state, its stability, and its folding pathway. To achieve this objective, an extensive NMR analysis will be made of the residual structure which persists in partially folded forms of nuclease. Preliminary work indicates that the amount and nature of this residual structure varies with the perturbation that causes break down of the folded, native state. Initial characterization of a low density denatured state, a large fragment designated delta131delta, will be systematically extended by fully assigning all of the side chain 1H and 13C resonances. An exhaustive search for side chain/side chain NOE's will involve the development of new pulse sequences that filter out intraresidue NOE's through the incorporation of specific 13C labelled amino acid residues and that transfer an NOE signal from side chain protons to the much more disperse backbone protons (H-alpha and H/N for detection. This structural analysis will be pursued to the highest level of resolution attainable. In order to quantitate the relative stability of different structural elements in delta131delta, NMR analysis will be undertaken in the presence of denaturants (e.g., urea) and stabilizers (e.g., glycerol). The dependence of these structural elements on other regions of the polypeptide chain will be investigated using both large peptides labeled with 15N/13C generated by chemical cleavage at uniquely introduced cysteine residues plus chemically synthesized small peptides. Refinement of a structural model of a highly compact """"""""quasi-native"""""""" form of nuclease will be pursued to high resolution, and the residual structure of a number of partially folded forms of nuclease induced by single substitution and insertion mutations will be analyzed and compared to the results obtained with delta131delta. The ultimate goal of this work is to construct an """"""""equilibrium folding pathway"""""""" for nuclease, one that represents the stabilities and the hierarchical interdependencies of the major chain/chain interactions. In effect, the """"""""free energy distance"""""""" between the denatured and native states becomes the axis along which structure develops, taking the place of time as the variable in the more conventional kinetic folding approach. And finally, empirical equations for calculating the stability effects of alanine and glycine substitutions in staph nuclease will be developed, refined, and tested by building upon the extensive mutant database already in hand and by employing mathematical techniques such as statistical factor analysis, neural networks and fuzzy logic.
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