The long term objective of this proposal is to gather enough empirical data in order to predict the properties of proteins from their amino acid sequence. This is to be done for a family of small, rigid protein proteinase inhibitors - the avian ovomucoid third domains. The work consists of two phases: production of variants and measurement of various bird egg whites, protein semisynthesis and site specific mutagenesis. It is anticipated that about 200 additional variants will be produced in the next grant period. The properties will be measured in part in this laboratory and in part by many collaborators throughout the world. As ovomucoid third domains often are potent inhibitors of serine proteinases their interaction with bovine alpha chymotrypsin, porcine pancreatic elastase, subtilisin Carlsberg and Streptomyces griseus proteinases A and B is studied. We measure the equilibrium constant for enzyme-inhibitor association and the rate constants characterizing the interaction. Collaborators determine their dimensional structure of enzyme-inhibitor complexes and of free inhibitors. For inhibitors alone we measure the reactive site peptide bond hydrolysis equilibrium constant, Khyd and the denaturation equilibrium constant Kden. Various spectroscopy (UV, laser Raman, fluorescence, NMR and mass spectral) measurements are carried out as well as antigenic can chromatographic characterizations. The sequence data are all used to construct phylogenies of birds and to understand the unusual evolutional property of protein proteinase inhibitors: the hypervariability of enzyme-inhibitor contact positions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM010831-32
Application #
3268149
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1977-09-01
Project End
1992-05-31
Budget Start
1991-04-01
Budget End
1992-05-31
Support Year
32
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Purdue University
Department
Type
Schools of Arts and Sciences
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907