title: Mapping temperature-induced conformational changes in the Escherichia coli heat shock transcription factor sigma 32 by amide hydrogen exchange.
abstract: Stress conditions such as heat shock alter the transcriptional profile in all organisms. In Escherichia coli the heat shock transcription factor, sigma 32, out-competes upon temperature up-shift the housekeeping sigma-factor, sigma 70, for binding to core RNA polymerase and initiates heat shock gene transcription. To investigate possible heat-induced conformational changes in sigma 32 we performed amide hydrogen (H/D) exchange experiments under optimal growth and heat shock conditions combined with mass spectrometry. We found a rapid exchange of around 220 of the 294 amide hydrogens at 37 degrees C, indicating that sigma 32 adopts a highly flexible structure. At 42 degrees C we observed a slow correlated exchange of 30 additional amide hydrogens and localized it to a helix-loop-helix motif within domain sigma 2 that is responsible for the recognition of the -10 region in heat shock promoters. The correlated exchange is shown to constitute a reversible unfolding with a half-life of about 30 min due to a temperature-dependent decrease in stabilization energy. We propose that this gradual decrease in stabilization energy of domain sigma 2 with increasing temperatures facilitates the unfolding of sigma 32 by the AAA+ protease FtsH thereby decreasing its half-life. Taken together our data show that the sigma 2 domain of sigma 32 can act as a thermosensor, which might be important for the heat shock regulation.
mesh_terms: D000577:Amides; D020816:Amino Acid Motifs; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006207:Half-Life; D006360:Heat-Shock Proteins / Q000737:chemistry*; D010449:Peptide Mapping; D011487:Protein Conformation; D020673:Protein Renaturation; D012808:Sigma Factor / Q000737:chemistry; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry*
chemical_list: D000577:Amides; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases