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J. Biol. Chem., Vol. 283, Issue 5, 2883-2895, February 1, 2008

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Conformational Stability of Helicobacter pylori Flavodoxin

FIT TO FUNCTION AT pH 5^*

Nunilo Cremades¹, Marta Bueno¹, José Luis Neira^¶, Adrián Velázquez-Campoy, and Javier Sancho²

From the Biocomputation and Complex Systems Physics Institute and the Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza and the ^¶Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche, Alicante, Spain

Flavodoxin is an essential protein for Helicobacter pylori, a pathogen living in the very acidic environment of the gastric tract and responsible for several diseases. We report the conformational stability of the protein in neutral and acidic pH. The apoprotein remains native between pH 12 and 5 and adopts a monomeric molten globule conformation at more acidic pH values. The equilibrium unfolding in urea appears two-state for either conformation, but the native one coexists with a hidden equilibrium intermediate of very similar properties. The stability of H. pylori apoflavodoxin is higher than that of the Anabaena homologue throughout the entire pH interval, which may be related to better charge compensation. H. pylori apoflavodoxin is strongly stabilized by its FMN cofactor. A global analysis of apo- and holoflavodoxin equilibrium unfolding, with and without excess FMN, indicates that the cofactor only binds to the native state. Some physical-chemical properties of the protein may represent an adaptation to the acidic environment. Unlike the apoflavodoxin from Anabaena, which becomes highly insoluble at pH 5.0, that from H. pylori remains soluble to at least 40 µM. This fact, together with the high stability of the apoprotein at this low pH that can arise in the bacteria cytoplasm, seems useful to allow newly synthesized apoflavodoxin molecules to fold and remain soluble to accomplish cofactor binding, which in turn increases the stability. Also, whenever the cytoplasmic pH drops to 5, preexisting flavodoxin molecules will remain folded and soluble and will retain the FMN cofactor, thus remaining functional.

Received for publication, July 11, 2007 , and in revised form, November 9, 2007.

^* This work was supported in part by Grants BFU2004-01411, CTQ2005-00360/BQU, and SAF2004-07722 from the Ministerio de Educación y Ciencia, Spain, and Grant PM076/2006 from the Gobierno de Aragón, Spain. 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.

¹ Supported by Ministerio de Educación y Ciencia fellowships (Spain).

² To whom correspondence should be addressed. Tel.: 34-976761286; Fax: 34-976762123; E-mail: jsancho{at}unizar.es.

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