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J. Biol. Chem., Vol. 279, Issue 12, 10955-10961, March 19, 2004

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Role of the Transmembrane Domain in the Stability of TrwB, an Integral Protein Involved in Bacterial Conjugation^*

Itsaso Hormaeche, Ibón Iloro, José L. R. Arrondo, Félix M. Goñi, Fernando de la Cruz¶, and Itziar Alkorta

From the Unidad de Biofísica (Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad del País Vasco), and Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain and ^¶Departamento de Biología Molecular, Universidad de Cantabria, 39011 Santander, Spain

TrwB is an integral membrane protein encoded by the conjugative plasmid R388. TrwB binds ATP and is essential for R388-directed bacterial conjugation. The protein consists of a cytosolic domain, which contains an ATP-binding site, and a transmembrane domain. The complete protein has been purified in the presence of detergents, and in addition, the cytosolic domain has also been isolated in the form of a soluble truncated protein, TrwBN70. The availability of intact and truncated forms of the protein provides a convenient system to study the role of the transmembrane domain in the stability of TrwB. Protein denaturation was achieved by heat, in the presence of guanidinium HCl, or under low salt conditions. In all three cases TrwB was significantly more stable than TrwBN70 with other conditions being the same. IR spectroscopy of the native and truncated forms revealed significant differences between them. In addition, it was found that TrwBN70 was stabilized in dispersions of non-ionic detergent, suggesting the presence of hydrophobic patches on the surface of the truncated protein. IR spectroscopy also confirmed the conformational stability provided by the detergent. These results suggest that in integral membrane proteins consisting of a transmembrane and a cytosolic domain, the transmembrane portion may have a role beyond the mere anchoring of the protein to the cell membrane. In addition, this study indicates that the truncated soluble parts of two-domain membrane proteins may not reflect the physiological conformation of their native counterparts.

Received for publication, September 22, 2003 , and in revised form, December 23, 2003.

^* This study was supported in part by grants from the Ministerio de Ciencia y Tecnología (BMC 2001-0791) and Universidad del País Vasco (13552/2001) (to F. M. G.) and from the Ministerio de Ciencia y Tecnología (BMC 2002-00379 to F. de la C.). 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. E-mail: gbpgourf{at}lg.ehu.es.

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