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J. Biol. Chem., Vol. 278, Issue 40, 38247-38253, October 3, 2003

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Antifolding Activity of the SecB Chaperone Is Essential for Secretion of HasA, a Quickly Folding ABC Pathway Substrate^*

Nicolas Wolff, Guillaume Sapriel, Christophe Bodenreider¶, Alain Chaffotte¶, and Philippe Delepelaire||

From the Unité de Résonance Magnétique Nucléaire des Biomolécules, the ^¶Unité de Repliement et Modélisation des Protéines, and the Unité des Membranes Bactériennes CNRS UMR2172, Institut Pasteur, 25–28 rue du Dr. Roux, 75724 Paris Cedex 15, France

We have previously shown that SecB, the ATP-independent chaperone of the Sec pathway, is required for the secretion of the HasA hemophore from Serratia marcescens via its type I secretion pathway, both in the reconstituted system in Escherichia coli and in the original host. The refolding of apo-HasA after denaturation with guanidine HCl was followed by stopped-flow measurements of fluorescence of its single tryptophan, both in the absence and presence of SecB. In the absence of SecB, HasA folds very quickly with one main phase (45 s^–1) accounting for 92% of the signal. SecB considerably slows down HasA folding. At stoichiometric amounts of SecB and HasA, a single phase (0.014 s^–1) of refolding is observed. Two double point mutants of HasA were made, abolishing two hydrogen bonds between N-terminal and C-terminal side chain residues. In both cases, the mutants essentially maintained the same secondary and tertiary structure as wild-type HasA and were fully functional. Refolding of both mutants was much slower than that of wild-type HasA and they were secreted essentially independently of SecB. We conclude that SecB has mainly an antifolding function in the HasA ABC secretion pathway.

Received for publication, March 6, 2003 , and in revised form, June 26, 2003.

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

^|| To whom correspondence should be addressed. Tel.: 33-1-40-61-32-76; Fax: 33-1-45-68-87-90; E-mail: pdelep{at}pasteur.fr.

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