The general goal of this project is to determine the internal motions of proteins by which atoms buried in the matrix of folded proteins are accessible to solvent. This is measured by the hydrogen isotope exchange kinetics of solvent hydrogen atoms with labile protein protons, most of which are peptide amide protons. The hydrogen isotope exchange kinetics of peptide NN's in folded proteins are distributed over 6-10 orders of magnitude. The most rapidly exchangeing NH's are on the surface, and the slowest exchanging NH's tend to be in buried sections of Beta -sheet. Although buried NH's exchange slower than model compounds, their finite exchange rates demonstrate that interior atoms in native proteins have some probability of being exposed to solvent. This immediately implies that the protein fluctuates to render tightly packed, buried atoms accessible to solvent. Recently our laboratory has made the exciting findings that the exchange rates of NH's on the surface of bovine pancreatic trypsin inhibitor (BPTI) vary from 3-fold to 2000-fold slower than model compounds, that their pHmin values vary over greater than 2 pH units, and that some have pHmin of less than 1. It has been commonly assumed that surface NH atoms (not H-bonded and with finite static accessibility) exchange with rates comparable to those in model peptides. The deviation in exchange rates and pHmin from model compounds then taken on special interest as a reflection of the dynamic structure of the protein-solvent interface.
The specific aims of this research proposal are 1) to characterize the hydrogen exchange kinetics of NH's in BPTI with intermediate exchange rates, 2) to measure the effect of trypsinogen and trypsinogen/Ile-Val binding on the exchange rates of BPTI NH's 3) to compare the exchange rate of individual BPTI NH's in solution with their exchange rates in the crystalline and powder forms, and 4) to measure the exchange kinetics of water buried in the BPTI-trypsin complex with solvent water.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026242-08
Application #
3273759
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1979-04-01
Project End
1990-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
8
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Arts and Sciences
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Li, Renhao; Battiste, John L; Woodward, Clare (2002) Native-like interactions favored in the unfolded bovine pancreatic trypsin inhibitor have different roles in folding. Biochemistry 41:2246-53
Battiste, John L; Li, Renhao; Woodward, Clare (2002) A highly destabilizing mutation, G37A, of the bovine pancreatic trypsin inhibitor retains the average native conformation but greatly increases local flexibility. Biochemistry 41:2237-45
Barbar, E; Hare, M; Makokha, M et al. (2001) NMR-detected order in core residues of denatured bovine pancreatic trypsin inhibitor. Biochemistry 40:9734-42
Woodward, C; Barbar, E; Carulla, N et al. (2001) Experimental approaches to protein folding based on the concept of a slow hydrogen exchange core. J Mol Graph Model 19:94-101
Li, R; Woodward, C (1999) The hydrogen exchange core and protein folding. Protein Sci 8:1571-90
Barbar, E (1999) NMR characterization of partially folded and unfolded conformational ensembles of proteins. Biopolymers 51:191-207
Akasaka, K; Li, H; Yamada, H et al. (1999) Pressure response of protein backbone structure. Pressure-induced amide 15N chemical shifts in BPTI. Protein Sci 8:1946-53
Barbar, E; Hare, M; Daragan, V et al. (1998) Dynamics of the conformational ensemble of partially folded bovine pancreatic trypsin inhibitor. Biochemistry 37:7822-33
Liang, J; Edelsbrunner, H; Woodward, C (1998) Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design. Protein Sci 7:1884-97
Barbar, E; LiCata, V J; Barany, G et al. (1997) Local fluctuations and global unfolding of partially folded BPTI detected by NMR. Biophys Chem 64:45-57

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