Protein-protein association is a central event in a diverse range of biological processes including enzyme catalysis/inhibition and electron transport. The orientational constraints for forming a stereospecific complex severely restrict the rate of association. Electrostatic interactions are able to compensate for such restriction and enhance the rate by as much as four orders of magnitude. Through a series of theoretical and computational studies, the principal investigator recently has shown that the electrostatic enhancement of the diffusion- influenced association rate can be predicted by the free energy of a """"""""transition state"""""""" for forming the stereospecific complex. To establish this as a truly predictive computational approach to protein-protein association, a structural model for the transition state proposed herein will be refined and validated against experimental results for the effects of charge mutations and ionic strength on several protein complexes. These include the barnaes-barstar complex, on which ongoing collaboration with Dr. Alan Fersht of the UK Medical Research Council will allow for predicted mutational effects to be tested. As applications, association and dissociation of cytochrome c and cytchrome c peroxidase will be studied to help settle the current debate about the rate-limiting step for electron transfer and the effects of mutations on the rate of hemoglobin AlphaBeta dimer assembly will be quantitatively determined to account for the relative proportions of normal and variant hemoglobins in red blood cells of heterozygotes. On a qualitative level, it is envisioned that the structural model for the transition state will serve as a guide for understanding the contributions of individual amino acids to protein-protein association.
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