We seek to characterize functional and conformational changes of the E. coli heat-shock protein GroEL at elevated temperatures. This investigation is important for understanding the function of this protein under heat-shock. The elevated expression of heat-shock proteins has been observed in cells and tissues in concert with a broad distribution of human diseases where symptoms include fever, inflammation, ischemia, oxidant injury, cardiac hypertrophy, and cell and tissue damage. Our working hypothesis is that the heat-shock protein GroEL plays an essential role in protecting other proteins against damage caused by stressful conditions, such as heat-shock. The broad long-term objective is to establish structure-function relationships of the heat shock protein GroEL under unfavorable cellular conditions.
The specific aims of this project are: 1) Characterization of the interaction between GroEL and enzymes undergoing denaturation at elevated temperatures. This will be achieved by examining mixtures of both a thermally-denatured enzyme and the GroEL protein using high-speed centrifugation and size exclusion HPLC. 2) Evaluation of the stability of complexes of GroEL and thermally- denatured enzymes. The integrity of the complexes over time will be monitored by high-speed centrifugation and size exclusion HPLC and by determining the activity of the released enzyme. 3) Analysis of the conformation of thermally-denatured enzymes bound to GroEL. This will be achieved by using fluorescence spectroscopy and limited proteolysis. 4) To detail the thermal unfolding of GroEL and the influence of its ligands on the thermal unfolding of the chaperonin. This will be achieved by using tyrosine and bis-ANS fluorescence spectroscopy, light scattering techniques and circular dichroism spectroscopy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Minority Biomedical Research Support - MBRS (S06)
Project #
5S06GM059833-02
Application #
6349132
Study Section
Minority Programs Review Committee (MPRC)
Project Start
2000-09-01
Project End
2001-08-31
Budget Start
Budget End
Support Year
2
Fiscal Year
2000
Total Cost
$184,123
Indirect Cost
Name
California State University San Marcos
Department
Type
DUNS #
176262681
City
San Marcos
State
CA
Country
United States
Zip Code
92078
Melkani, Girish C; Sielaff, Robin; Zardeneta, Gustavo et al. (2012) Interaction of oxidized chaperonin GroEL with an unfolded protein at low temperatures. Biosci Rep 32:299-303
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Melkani, Girish C; Zardeneta, Gustavo; Mendoza, Jose A (2005) On the chaperonin activity of GroEL at heat-shock temperature. Int J Biochem Cell Biol 37:1375-85

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