The broad, long-term objective of this work is to elucidate the rules that govern protein folding. Bovine pancreatic trypsin inhibitor (BPTI) was chosen as a model system because it is a simple globular polypeptide and a large amount of structural, biophysical, and theoretical data is already available for the wild type protein. BPTI is also the best understood member of the Kunitz class of protease inhibitors, a family that includes several members relevant to human physiology and disease including the lipoprotein-associated coagulation inhibitor and the alternatively-spliced domain of the Alzheimer's amyloid beta protein precursor. The overall basic thrust of the research has been to make genetically-modified forms of BPTI that have perturbed folding properties, express these proteins in quantity, and characterize them biophysically. The specific research priorities for this grant are: 1) Address the problem of """"""""designing out"""""""" disulfide bonds by making BPTI mutants missing two or more of the native disulfides; 2) Conduct a quantitative survey of all stabilizing interactions in the molecule by systematically mutating each non-alanine residue to alanine and characterizing the stabilization free energies of the resulting mutants; 3) Test the """"""""cardboard box"""""""" model of protein folding by measuring the contribution of electrostatics to 5-55 disulfide bond formation and to global polypeptide stability; 4) Facilitate ongoing work in collaborators' laboratories aimed at investigating the structure and dynamics of selected BPTI mutants. Presumably, this combined genetic and biophysical approach to the dissection of the folding mechanism of a very simple model protein will illuminate the still poorly-understood process of protein folding in general.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
1R01AG010462-01
Application #
3122364
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1991-07-01
Project End
1994-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Rutgers University
Department
Type
Organized Research Units
DUNS #
038633251
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901
Qasim, M A; Lu, Wuyuan; Lu, Stephen M et al. (2003) Testing of the additivity-based protein sequence to reactivity algorithm. Biochemistry 42:6460-6
Lu, W; Apostol, I; Qasim, M A et al. (1997) Binding of amino acid side-chains to S1 cavities of serine proteinases. J Mol Biol 266:441-61
Liu, Y; Breslauer, K; Anderson, S (1997) ""Designing out"" disulfide bonds: thermodynamic properties of 30-51 cystine substitution mutants of bovine pancreatic trypsin inhibitor. Biochemistry 36:5323-35
Kim, S; Baum, J; Anderson, S (1997) Production of correctly folded recombinant [13C, 15N]-enriched guinea pig [Val90]-alpha-lactalbumin. Protein Eng 10:455-62
Castro, M J; Anderson, S (1996) Alanine point-mutations in the reactive region of bovine pancreatic trypsin inhibitor: effects on the kinetics and thermodynamics of binding to beta-trypsin and alpha-chymotrypsin. Biochemistry 35:11435-46
Jansson, M; Li, Y C; Jendeberg, L et al. (1996) High-level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli. J Biomol NMR 7:131-41
Abul Qasim, M; Ranjbar, M R; Wynn, R et al. (1995) Ionizable P1 residues in serine proteinase inhibitors undergo large pK shifts on complex formation. J Biol Chem 270:27419-22
Kingston, I B; Castro, M J; Anderson, S (1995) In vitro stimulation of tissue-type plasminogen activator by Alzheimer amyloid beta-peptide analogues. Nat Med 1:138-42
Castro, M J; Kingston, I B; Anderson, S (1995) A spectrophotometric assay for the determination of the catalytic efficiency of plasminogen activators using a slowly hydrolyzed plasmin substrate. Anal Biochem 226:225-31
Lu, W; Zhang, W; Molloy, S S et al. (1993) Arg15-Lys17-Arg18 turkey ovomucoid third domain inhibits human furin. J Biol Chem 268:14583-5

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