INVESTIGADORES
RODRIGUEZ Fernanda Mariana
congresos y reuniones científicas
Título:
Identification of the DnaK binding site and of chaperone-induced unfolding within the heat shock transcription factor sigma32.
Autor/es:
RODRIGUEZ FERNANDA; RIST WOLFGANG; MAYER MATTHIAS P; BUKAU BERND
Lugar:
Blaubeuren
Reunión:
Simposio; 25th Symposium on Mechanisms of Gene Regulation; 2005
Resumen:
The heat-shock response is a protective mechanism of cells against stress-induced damage of protein. In E. coli this response is mediated by the alternative sigma transcription factor sigma32 which activates the transcription of the heat-shock genes. Stress-dependent changes in heat-shock gene expression are mediated by changes in the activity and stability of sigma32. The DnaK system plays an important role in the regulation of the heat-shock response, because its association with sigma32 at optimal growth temperatures prevents the formation of the RNA polymerase-sigma32 complex. Hence, the expression of heat-shock proteins is determined by a homeostatic balance between DnaK bound to denatured proteins and DnaK interacting with sigma32. The DnaK chaperone system acts as a negative modulator of the heat-shock response by regulating the stability and the activity of sigma32. However, the precise mechanism of the DnaK interaction with sigma32 is still unclear. In order to get a complete picture of the heat-shock regulation, it is important to characterize the regions of sigma32 directly involved in the DnaK-mediated control. We identified the DnaK binding site within sigma32. Protease footprinting revealed a DnaK-protected site at residues 198-204 of sigma32. This site is located within the only major segment of sigma32 predicted to be unstructured. Mutational alterations of the site decreased the affinity of sigma32 for DnaK. This binding site was confirmed using H/D footprinting. In analogy to protease footprinting, H/D footprinting can give information about binding interfaces in protein complexes. Amide hydrogen exchange was performed with sigma32 alone and in complex with DnaK. Two peptides, residues 183-199 and 200-208, showed protection when bound to DnaK whereas all others did not. As a positive control we used a peptide substrate of DnaK (sigma32 M195-N207) which shows a similar behaviour. Furthermore, amide hydrogen exchange revealed a region that incorporated more hydrogens in the presence of DnaK than in its absence. This indicates a chaperone-induced unfolding of a secondary structure element that may facilitate degradation of sigma32 in vivo.