The long-term goals of this application are to understand the structural origins of the catalytic power of enzymes, to understand the functional importance of protein conformational changes and atomic fluctuations, to characterize the structural consequences of radiation damage in proteins and nucleic acids, and to apply the techniques of protein crystallography and computer modelling to provide a detailed molecular picture of the bacterial chemotactic sensory system. The first of these aims should provide basic information important to rational design of enzyme-active drugs and to an understanding of several diseases, such as emphysema, that involve enzyme deficiency or where an enzyme is the critical toxic substance. Knowledge of the changes in structure that proteins undergo both in response to the binding of specific ligands and as a consequence of the kinetic energy available at ordinary temperatures will be complementary to the structural picture of the static protein. Radiation damage to living organisms is a significant health consideration, and the precise details of the structures of the damaged molecules are often uncertain. Characterization of the structures of the damaged proteins and nucleic acids should aid in the design of radiation-protecting compounds, and also may be of use for people who consider the use of radiation as a food preservative measure. The bacterial chemotactic system is a good potential model for the sensory systems in human beings, such as smell, taste and possibly touch.
Our specific aims i nclude the determination of the three-dimensional structure of B. stearothermophilus alanine racemase at 2.5A resolution or higher by the method of single-crystal X-ray diffraction, the determination of the structures of the complex of this enzyme with substrate D- or L- alanine and with a phosphonate slow-binding inhibitor; the crystallization and crystal structure determination of the methyl esterase from the chemotactic system of Salmonella typhimurium; the determination and refinement at high resolution (i.e., about 1.5A) of the crystal structures of elastase complexed with substrate in its acyl-enzyme intermediate form, the enzyme-product form, and the Michaelis complex of enzyme with substrate prior to acylation by low-temperature X-ray crystallography; characterization of the structure of sperm-whale met-myoglobin at 50% radiation damage; characterization of several DNA-like oligonucleotide duplex molecules at similar doses, and the complete determination of the pressure and temperature dependence of two protein structures.
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