The long term objective of this grant is to advance fundamental knowledge of the mechanism of protein hydrogen exchange, a unique and versatile probe of protein dynamic structure. Hydrogen exchange in proteins occurs either by internal motions of the folded state, or by global cooperative unfolding, and we call this the two-process model of exchange. To advance understanding of each process, we characterized relationships between hydrogen exchange rates and average (crystal) structure of the folded state, as well as relationships between hydrogen exchange rates and global unfolding. Toward this end, the work funded by this grant since the last competitive renewal produced a significant body of data on dynamics, folding thermodynamics and crystal structure for 8 variants of the small model protein, bovine pancreatic trypsin inhibitor (BPTI). The research plan in this proposal is twofold; i) to bring the data base we have built to bear on further important questions of mechanism of hydrogen exchange in proteins and of the relationship of dynamics measured by hydrogen exchange to protein average structure (crystal or NMR) and to protein folding, and ii) to test our hypothesis relating protein dynamic structure to protein folding, namely, that the native state slow exchange core is the folding core.
Specific aims i nclude the following. a) Exchange rates of surface NHs in BPTI mutants will be determined. b) Double mutant combinations of our single-site mutants will be constructed in order to examine BPTI denaturation at low pH. c) Highly accurate thermodynamics for the cooperative folding transition of single site BPTI mutants will be obtained in collaboration with P. Privalov. d) Pulsed exchange experiments on BPTI mutants will be carried out. e) NMR and hydrogen exchange experiments will be carried out on BPT1 analog [14-38]Abu which retains the 14-38 disulfide bond but has the other four cysteines replaced by alpha-amino butyric acid. We will characterize the hydrogen exchange properties, folding thermodynamics and NMR dynamic structure of [14-18]Abu at pH 4.5-6 and 3-10 degree C, conditions under which it is an ensemble of partially folded structures, and a model for early intermediates in BPTI folding. Other NMR experiments are planned for variants of [14-38]Abu under partially folding conditions, and for [14-38]Abu at pH less then 2 and 5 degree C, conditions under which it forms an acid state (A state). f) NMR structures and hydrogen exchange of two analogs of reduced BPTI, models for the unfolded state of the protein, will be characterized. Preliminary evidence strongly suggest hydrophobic clustering and non-random, possibly non-local, NMR-detected structure in reduced BPTI.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026242-20
Application #
2838454
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1979-04-01
Project End
2000-09-30
Budget Start
1998-12-01
Budget End
2000-09-30
Support Year
20
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Biochemistry
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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