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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biophysical Chemistry Study Section (BBCB)
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University of California San Francisco
Schools of Pharmacy
San Francisco
United States
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Eigenbrot, C; Randal, M; Kossiakoff, A A (1992) Structural effects induced by mutagenesis affected by crystal packing factors: the structure of a 30-51 disulfide mutant of basic pancreatic trypsin inhibitor. Proteins 14:75-87
Kossiakoff, A A; Sintchak, M D; Shpungin, J et al. (1992) Analysis of solvent structure in proteins using neutron D2O-H2O solvent maps: pattern of primary and secondary hydration of trypsin. Proteins 12:223-36
Kossiakoff, A A; Ultsch, M; White, S et al. (1991) Neutron structure of subtilisin BPN': effects of chemical environment on hydrogen-bonding geometries and the pattern of hydrogen-deuterium exchange in secondary structure elements. Biochemistry 30:1211-21
Hynes, T R; Randal, M; Kennedy, L A et al. (1990) X-ray crystal structure of the protease inhibitor domain of Alzheimer's amyloid beta-protein precursor. Biochemistry 29:10018-22
Kossiakoff, A A; Shpungin, J; Sintchak, M D (1990) Hydroxyl hydrogen conformations in trypsin determined by the neutron diffraction solvent difference map method: relative importance of steric and electrostatic factors in defining hydrogen-bonding geometries. Proc Natl Acad Sci U S A 87:4468-72
Katz, B; Kossiakoff, A A (1990) Crystal structures of subtilisin BPN' variants containing disulfide bonds and cavities: concerted structural rearrangements induced by mutagenesis. Proteins 7:343-57
Eigenbrot, C; Randal, M; Kossiakoff, A A (1990) Structural effects induced by removal of a disulfide-bridge: the X-ray structure of the C30A/C51A mutant of basic pancreatic trypsin inhibitor at 1.6 A. Protein Eng 3:591-8