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Projekt
Structure-Function Analysis of the Heat Shock Protein DegP
Antragsteller
Privatdozent Dr. Tim Clausen
Fachliche Zuordnung
Stoffwechselphysiologie, Biochemie und Genetik der Mikroorganismen
Förderung
Förderung von 2002 bis 2005
Projektkennung
Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 5361566
Proteases and chaperones together serve to maintain quality control of cellular proteins. The combination of chaperone and protease function in a single protein could provide a direct and rapid response to protein folding problems or the need to adjust protein concentrations. The heat shock protein DegP (HtrA, Protease Do) exhibits these dual functionalities and thus offers unique possibilities to investigate how cells distinguish between proteins that can be refolded and "hopeless" cases that need to be degraded. DegP is a widely conserved protein found in most organisms including humans. Initially identified as an energy-independent serine protease, it is involved in tolerance against folding stress and in virulence of pathogenic bacteria. Mature DegP from Escherichia coli, a hexamer of 300 kDa, has 448 residues per subunit of which His105, Asp135 and Ser210 form the catalytic triad. At its C-terminus, DegP has two PDZ domains, which are believed to mediate protein-protein interaction. As mentioned before, DegP combines both general chaperon and proteolytic functions. Interestingly, the chaperon function dominates at low temperature, while the switch to proteolytic activity was observed at elevated temperatures. In the last two years, we improved the purification of DegP from E. coli to such an extent that the proteolytically inactive mutant S210A, in which the active site serine was replaced by alanine, could be crystallized. The crystals diffract to 2.6 Å resolution, and the structure solution process is almost finished. We succeeded to localize the Se position in SeMet derivatized crystals and carried out a Multiple Anomalous Diffraction experiment. With the applied funding we plan to perform a detailed structure-function analyses of DegP from E. coli and to extend this approach to related proteins from other organisms. The results are expected to provide novel insights how a single cellular factor can channel different substrates into one of the two key pathways controlling protein stability and protein turnover.
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