The broad, long term objectives of this research are to develop and implement new ways of determining and refining protein structures, primary in solution but also in the solid-state. The health relatedness of this research is two-fold. First, with the availability of higher-resolution protein structures in solution it will begin to be possible to apply quantum chemical techniques to solving problems such as the mechanisms of actions of enzymes, and the interactions of drugs with suitable target molecules. Second, many proteins exist and function in the solid-state but their structure are only just beginning to be determined at low resolution. The chemical shift tensors and their orientations determined in this research will facilitate further solid-state structure refinements and determinations.
The specific aims are therefore: 1) to finalize the computation of the carbon-13 NMR shielding surfaces for the abundant conformers of the naturally occurring amino-acids; 2) to analyze the C/alpha shielding tensor information now becoming available for proteins in solution; 3) to incorporate both chemical shift, chemical shift anisotropy and dipolar splitting information into structure refinements of proteins (including ubiquitins, interleukin-8, Staphylococcal nuclease and hen egg white lysozyme); 4) to investigate the possibilities of determining protein structures in solution using primarily chemical shift, shift anisotropy an dipolar splitting information; 5) to determine experimentally and computer theoretically 13C, 15N and 17O shifts and shielding tensors in model systems having well-defined structures (to validate shielding tensor calculations), 6) to investigate hydrogen bonding in model systems using a combination of 1H, 2H, 13C, 15N and 17O NMR shift anisotropy and where appropriate electric field gradient tensor information, with particular emphasis on analyzing the effects of bond length and in-plane and out-of-plane hydrogen bond angles on the spectroscopic observables; and finally, 7) to then apply these techniques to obtain more detailed information on hydrogen bonding in proteins in solution. Overall, the basic aim is to combine chemical shifts, chemical shift anisotropies, electric field gradient tensor and dipolar splitting information to refine the structures of proteins in solution, and to further developing the chemical shift/chemical shielding tensor approach to structure refinement and determination in the solid-state.
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