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J. Biol. Chem., Vol. 278, Issue 51, 51415-51421, December 19, 2003

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Mapping Temperature-induced Conformational Changes in the Escherichia coli Heat Shock Transcription Factor ³² by Amide Hydrogen Exchange^*

Wolfgang Rist, Thomas J. D. Jørgensen, Peter Roepstorff, Bernd Bukau¶, and Matthias P. Mayer¶

From the Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany and the Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark

Stress conditions such as heat shock alter the transcriptional profile in all organisms. In Escherichia coli the heat shock transcription factor, ³², out-competes upon temperature up-shift the housekeeping -factor, ⁷⁰, for binding to core RNA polymerase and initiates heat shock gene transcription. To investigate possible heat-induced conformational changes in 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 °C, indicating that ³² adopts a highly flexible structure. At 42 °C we observed a slow correlated exchange of 30 additional amide hydrogens and localized it to a helix-loop-helix motif within domain 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 ₂ with increasing temperatures facilitates the unfolding of ³² by the AAA+ protease FtsH thereby decreasing its half-life. Taken together our data show that the ₂ domain of ³² can act as a thermosensor, which might be important for the heat shock regulation.

Received for publication, July 3, 2003 , and in revised form, August 28, 2003.

^* This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB388, SFB352, Leibnizprogramm) (to B. B. and M. P. M.) and the Fonds der Chemischen Industrie (Kékulé scholarship) and Marie Curie Short Term Fellowship (to W. R.), and DABIC (Danish Biotechnology Instrument Center) (to P. R. and T. J. D. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence may be addressed: Zentrum für Molekulare Biologie Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany. Tel.: 49-6221-546829; Fax: 49-6221-545894; E-mail: M. Mayer{at}zmbh.uni-heidelberg.de or bukau{at}zmbh.uni-heidelberg.de.

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