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J. Biol. Chem., Vol. 280, Issue 34, 30063-30072, August 26, 2005

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The YoeB Toxin Is a Folded Protein That Forms a Physical Complex with the Unfolded YefM Antitoxin

IMPLICATIONS FOR A STRUCTURAL-BASED DIFFERENTIAL STABILITY OF TOXIN-ANTITOXIN SYSTEMS^*

Izhack Cherny, Liat Rockah, and Ehud Gazit

From the Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

The chromosomal YoeB-YefM toxin-antitoxin module common to numerous strains of bacteria is presumed to have a significant role in survival under stringent conditions. Recently we showed that the purified YefM antitoxin is a natively unfolded protein, as we previously reported for the Phd antitoxin in the P1 phage Doc-Phd toxin-antitoxin system. Here we report the purification and structural properties of the YoeB toxin and present physical evidence for the existence of a tight YoeB·YefM polypeptide complex in solution. YoeB and YefM proteins co-eluted as single peaks in sequential Ni-affinity FPLC and Q-Sepharose ion-exchange chromatography implying the formation of a YoeB·YefM complex. The unstable antitoxin was removed from the mixture by natural proteolysis, and the residual YoeB protein was purified using ion exchange chromatography. Fluorescence anisotropy studies of the purified YoeB and YefM proteins showed a 2:1 stoichiometry of the complex, providing direct evidence for a physical complex between the proteins. Near- and far-UV circular dichroism spectroscopy of the purified toxin revealed that, similar to the Doc toxin, YoeB is a well-folded protein. Thermal denaturation experiments confirmed the conformational stability of the YoeB toxin, which underwent reversible thermal unfolding at temperatures up to 56 °C. The thermodynamic features of the toxin-antitoxin complex were similar. Taken together, our results support the notion of a correlation between differential physiological and structural stability in toxin-antitoxin modules.

Received for publication, June 8, 2005 , and in revised form, June 23, 2005.

^* This research was supported in part by the European Union Contract QLK3-CT-2001-00277. 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 should be addressed: Dept. of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: 972-3-640-9030; Fax: 972-3-640-5448; E-mail: ehudg{at}post.tau.ac.il.

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