The aim of this investigation is to provide an experimental basis to better understand the nature of protein-protein and protein-solvent interactions. An experimental approach is proposed that couples the capabilities of protein engineering and crystallographic techniques, particularly neutron diffraction. The protein, subtilisin, has been genetically engineered to alter, in a controlled way, several physicochemical properties related to structural stability. A number of mutant subtilisins have been crystallized. These variant proteins will be studied crystallographically and biophysically and the resulting structural perturbations will be evaluated to identify systematic relationships between altered chemical features of the wild type and mutant molecules. Neutron diffraction coupled to the H/D exchange technique will be used to study the conformational dynamics of subtilisin. Differences in H/D exchange between various mutants will be measured. These data, combined with the sites location in the 3-D structure, will provide details of the nature of alterations in the breathing patterns existing between variant proteins. Because changes in dynamic properties correlate to changes in stabbility, this information is crucial to sorting out the relative stabilities and interdependence of structural domains in proteins and is inaccessible by other techniques. Changes in the chemical environment around mutation sites will be analyzed by determining perturbations in local water structure. Systematic relationships between observed changes and the geometric and chemical properties of the substituted amino acid side chain will be defined. D20-H20 solvent difference maps, a procedure unique to neutron diffraction, will be used to identify small changes in ordered and partially ordered solvent.
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