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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM035976-11
Application #
2178173
Study Section
Special Emphasis Panel (NSS)
Project Start
1985-06-01
Project End
1998-05-31
Budget Start
1995-06-01
Budget End
1996-05-31
Support Year
11
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Rotsaert, Frederik J; Pikus, Jeremie D; Fox, Brian G et al. (2003) N-isotope effects on the Raman spectra of Fe(2)S(2) ferredoxin and Rieske ferredoxin: evidence for structural rigidity of metal sites. J Biol Inorg Chem 8:318-26
Assadi-Porter, F M; Aceti, D J; Markley, J L (2000) Sweetness determinant sites of brazzein, a small, heat-stable, sweet-tasting protein. Arch Biochem Biophys 376:259-65
Assadi-Porter, F M; Aceti, D J; Cheng, H et al. (2000) Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin. Arch Biochem Biophys 376:252-8
Xia, B; Jenk, D; LeMaster, D M et al. (2000) Electron-nuclear interactions in two prototypical [2Fe-2S] proteins: selective (chiral) deuteration and analysis of (1)H and (2)H NMR signals from the alpha and beta hydrogens of cysteinyl residues that ligate the iron in the active sites of human ferredo Arch Biochem Biophys 373:328-34
Chae, Y K; Abildgaard, F; Royer, C A et al. (1999) Oligomerization of the EK18 mutant of the trp repressor of Escherichia coli as observed by NMR spectroscopy. Arch Biochem Biophys 371:35-40
Xia, B; Pikus, J D; Xia, W et al. (1999) Detection and classification of hyperfine-shifted 1H, 2H, and 15N resonances of the Rieske ferredoxin component of toluene 4-monooxygenase. Biochemistry 38:727-39
Qasim, M A; Lu, S M; Ding, J et al. (1999) Thermodynamic criterion for the conformation of P1 residues of substrates and of inhibitors in complexes with serine proteinases. Biochemistry 38:7142-50
Alam, S L; Volkman, B F; Markley, J L et al. (1998) Detailed NMR analysis of the heme-protein interactions in component IV Glycera dibranchiata monomeric hemoglobin-CO. J Biomol NMR 11:119-33
Prehoda, K E; Mooberry, E S; Markley, J L (1998) Pressure denaturation of proteins: evaluation of compressibility effects. Biochemistry 37:5785-90
Weber-Main, A M; Hurley, J K; Cheng, H et al. (1998) An electrochemical, kinetic, and spectroscopic characterization of [2Fe-2S] vegetative and heterocyst ferredoxins from Anabaena 7120 with mutations in the cluster binding loop. Arch Biochem Biophys 355:181-8

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