Mapping temperature-induced conformational changes in the Escherichia coli heat shock transcription factor sigma 32 by amide hydrogen exchange.
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.
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*
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