Protein folding in vivo is now understood to be assisted by a special class of helper proteins collectively known as chaperones. The work proposed in this grant will concentrate on the family of 70 kDa heat shock proteins (Hsp70), which is conserved from bacteria to mammals. The proteins are involved in the earliest stages of peptide translation and bind to nascent peptide chains and peptide chains destined for transport to cellular organelles. In addition, these proteins bind to proteins damaged by thermal and other stress (heat shock) or mutation. In this grant application, we propose to determine the three-dimensional structures and the dynamics of the peptide binding domains of two eukaryotic Hsp 70 proteins, the Hsc found in the cytosol and the BiP protein of the endoplasmic reticulum. In addition, the structures of complexes of these proteins with small peptides will be studied. Multi- nuclear, multi-dimensional NMR methods will be used; the information will be correlated with function using site-directed mutagenesis in a collaborative fashion. The objective of these studies is to gain insight in the basis of the function of these chaperone proteins. With this information, we may infer and rationalize the apparent specificity of the chaperones for hydrophobic peptides. We will also gain insight in possible conformational changes that modulate the affinity of Hsp70s for their targets. The structures of the bound peptides will indicate their mode of binding and will help resolve the question whether the Hsp70s are merely solubilizing unfolded proteins or if initial stages of protein folding are taking place in their binding cleft. The dynamic information will be integral part of the interpretation processes. Protein folding is very basic to cell functioning; it is only with properly and timely folding proteins that the cell can perform its complicated regulatory functions and growth cycles. The insight in the structural basis of in vivo protein folding that will be gained from the proposed studies is therefore of relevance to the understanding of these growth cycles and their disturbances.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052421-03
Application #
2415293
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1995-05-01
Project End
1999-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
3
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Physiology
Type
Organized Research Units
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Yip, Grover N B; Zuiderweg, Erik R P (2004) A phase cycle scheme that significantly suppresses offset-dependent artifacts in the R2-CPMG 15N relaxation experiment. J Magn Reson 171:25-36
Cai, Sheng; Stevens, Shawn Y; Budor, Andrew P et al. (2003) Solvent interaction of a Hsp70 chaperone substrate-binding domain investigated with water-NOE NMR experiments. Biochemistry 42:11100-8
Hinton, Ayana; Zuiderweg, Erik R P; Ackerman, Sharon H (2003) A purified subfragment of yeast Atp11p retains full molecular chaperone activity. J Biol Chem 278:34110-3
Kern, Dorothee; Zuiderweg, Erik R P (2003) The role of dynamics in allosteric regulation. Curr Opin Struct Biol 13:748-57
Shao, Weiping; Im, Sang-Choul; Zuiderweg, Erik R P et al. (2003) Mapping the binding interface of the cytochrome b5-cytochrome c complex by nuclear magnetic resonance. Biochemistry 42:14774-84
Stevens, Shawn Y; Cai, Sheng; Pellecchia, Maurizio et al. (2003) The solution structure of the bacterial HSP70 chaperone protein domain DnaK(393-507) in complex with the peptide NRLLLTG. Protein Sci 12:2588-96
Chung, Duane A; Zuiderweg, Erik R P; Fowler, Carol B et al. (2002) NMR structure of the second intracellular loop of the alpha 2A adrenergic receptor: evidence for a novel cytoplasmic helix. Biochemistry 41:3596-604
Zuiderweg, Erik R P (2002) Mapping protein-protein interactions in solution by NMR spectroscopy. Biochemistry 41:1-7
Khandelwal, P; Keliikuli, K; Smit, C L et al. (2001) 1H, 15N and 13C assignments of the N-terminal domain of Yersinia outer protein H in its apo form and in complex with a phosphotyrosine peptide. J Biomol NMR 21:69-70
Hall, D A; Vander Kooi, C W; Stasik, C N et al. (2001) Mapping the interactions between flavodoxin and its physiological partners flavodoxin reductase and cobalamin-dependent methionine synthase. Proc Natl Acad Sci U S A 98:9521-6

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