The 70 kilodalton heat shock-related proteins (HSP70-related) are wide- spread and essential for cell viability. They appear to be intimately involved the processes of protein folding, targeting and maturation. Bacterial and parasite HSP70-related proteins are highly immunogenic; host immune responses to these highly conserved proteins may lead to autoimmunity syndromes. This proposal focuses on the question: What are the biochemical functions of the HSP70-related proteins in cells? The general strategy is structure- function studies at the molecular level.
The specific aims are: 1.Elucidate the ATPase mechanism using x-ray crystallography and site- specific mutagenesis. 2.Search for evidence for or against a major conformational change in the proteins using small-angle solution x-ray scattering. 3.Delimit the substrate binding functions within the substrate recognition domain of the bovine heat shock cognate protein (HSC70), using clathrin as a substrate, by constructing (i) C-terminal deletion mutants and (ii) proteins which are chimeras of HSC70 and another HSP70 related protein, BiP. 4.Probe the functional significance of the structural and sequence similarities of the ATPase fragment and actin, using site-specific mutagenesis in conjunction with ATPase and clathrin uncoating activity assays. 5.Examine the feasibility of using Laue diffraction on the HSC70 ATPase fragment to determine the structures of the reaction intermediated during catalysis. 6.Attempt to crystallize intact HSP70-related proteins and/or their substrate recognition domains.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM039928-06
Application #
3297218
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1988-10-01
Project End
1995-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
6
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Stanford University
Department
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Xu, Xiaohua; Wang, Shuying; Hu, Yao-Xiong et al. (2007) The periplasmic bacterial molecular chaperone SurA adapts its structure to bind peptides in different conformations to assert a sequence preference for aromatic residues. J Mol Biol 373:367-81
Bitto, Eduard; McKay, David B (2004) Binding of phage-display-selected peptides to the periplasmic chaperone protein SurA mimics binding of unfolded outer membrane proteins. FEBS Lett 568:94-8
Kwon, Ae-Ran; Trame, Christine B; McKay, David B (2004) Kinetics of protein substrate degradation by HslUV. J Struct Biol 146:141-7
Trame, Christine B; McKay, David B (2003) Structure of the Yersinia enterocolitica molecular-chaperone protein SycE. Acta Crystallogr D Biol Crystallogr 59:389-92
Kwon, Ae-Ran; Kessler, Benedikt M; Overkleeft, Herman S et al. (2003) Structure and reactivity of an asymmetric complex between HslV and I-domain deleted HslU, a prokaryotic homolog of the eukaryotic proteasome. J Mol Biol 330:185-95
Bitto, Eduard; McKay, David B (2003) The periplasmic molecular chaperone protein SurA binds a peptide motif that is characteristic of integral outer membrane proteins. J Biol Chem 278:49316-22
Bitto, Eduard; McKay, David B (2002) Crystallographic structure of SurA, a molecular chaperone that facilitates folding of outer membrane porins. Structure 10:1489-98
Sousa, Marcelo C; Kessler, Benedikt M; Overkleeft, Herman S et al. (2002) Crystal structure of HslUV complexed with a vinyl sulfone inhibitor: corroboration of a proposed mechanism of allosteric activation of HslV by HslU. J Mol Biol 318:779-85
Wedekind, J E; Trame, C B; Dorywalska, M et al. (2001) Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity. J Mol Biol 314:823-37
Sousa, M C; McKay, D B (2001) Structure of Haemophilus influenzae HslV protein at 1.9 A resolution, revealing a cation-binding site near the catalytic site. Acta Crystallogr D Biol Crystallogr 57:1950-4

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