The objectives of this research project are to develop new methods for studying protein structure-function relationships in solution and to apply these to the solution of health-related problems in protein chemistry. Techniques to be developed include the following: (1) stable isotope labeling strategies and heteronuclear two-dimensional NMR methods for extensive sequence-specific assignments in larger proteins and protein- inhibitor complexes, (2) computer-assisted post-acquisition processing and analysis of two-dimensional NMR data from proteins, and (3) improved methods for determining local mobility in proteins. Problems in protein chemistry to be addressed include: (1) experimentally testing whether compact units identified within proteins correspond to structural domains, (2) investigation of effects of single amino acid substitutions on the relative stability and dynamics of a series of proteinase inhibitors (avian ovomucoid third domains), (3) investigation of effects of amino acid substitutions on the energies of conformational substrates of a native and denatured protein (staphylococcal nuclease), (4) the mechanism of inhibition of human leukocyte elastase by peptide and protein inhibitors, and (5) design and testing of improved elastase inhibitors based on a series of amino acid replacements starting with turkey ovomucoid third domain. Insofar as possible, the proteins to be studied will be cloned and overproduced in E. coli to facilitate large-scale (100-mg) protein production and biosynthetic incorporation of stable isotopes. Multinuclear one- and two-dimensional NMR spectroscopy will be carried out at 9.40, 11.7, and 14.1 Tesla. Solution structures of the proteins, based on measurements of site-specific nuclear Overhauser enhancements and coupling constants, will be determined where possible by distance-geometry and/or restrained molecular dynamics. The results will be compared with available information from single-crystal diffraction studies. Although single amino acid modifications underlie many genetic diseases and cellular transformations, their effects are not well understood. Disruptions in the balance of elastase and its inhibitors is thought to lead to tissue damage as observed in chronic diseases such as adult respiratory distress syndrome, cystic fibrosis, pulmonary emphysema, and rheumatoid arthritis as well as clotting disorders; fundamental information about the mechanism of elastase inhibitors should be of utility in the design of improved therapeutic molecules.
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