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J. Biol. Chem., Vol. 279, Issue 45, 47184-47191, November 5, 2004

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The Long and Short Flavodoxins

II. THE ROLE OF THE DIFFERENTIATING LOOP IN APOFLAVODOXIN STABILITY AND FOLDING MECHANISM^*

Jon López-Llano¶, Susana Maldonado¶, Shandya Jain||**, Anabel Lostao, Raquel Godoy-Ruiz, José M. Sanchez-Ruiz, Manuel Cortijo||, Juan Fernández-Recio¶¶, and Javier Sancho||||

From the Biocomputation and Complex Systems Physics Institute, Zaragoza University, Zaragoza, Spain, Departamento Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza 50009, Zaragoza, Spain, ^||Instituto de Estudios Biofuncionales, Universidad Complutense de Madrid, Madrid, Spain, and Facultad de Ciencias, Departamento de Química Fisica, Universidad de Granada, 18071 Granada, Spain

Flavodoxins are classified in two groups according to the presence or absence of a 20-residue loop of unknown function. In the accompanying paper (36), we have shown that the differentiating loop from the long-chain Anabaena PCC 7119 flavodoxin is a peripheral structural element that can be removed without preventing the proper folding of the apoprotein. Here we investigate the role played by the loop in the stability and folding mechanism of flavodoxin by comparing the equilibrium and kinetic behavior of the full-length protein with that of loop-lacking, shortened variants. We show that, when the loop is removed, the three-state equilibrium thermal unfolding of apoflavodoxin becomes two-state. Thus, the loop is responsible for the complexity shown by long-chain apoflavodoxins toward thermal denaturation. As for the folding reaction, both shortened and wild type apoflavodoxins display three-state behavior but their folding mechanisms clearly differ. Whereas the full-length protein populates an essentially off-pathway transient intermediate, the additional state observed in the folding of the shortened variant analyzed seems to be simply an alternative native conformation. This finding suggests that the long loop may also be responsible for the accumulation of the kinetic intermediate observed in the full-length protein. Most revealing, however, is that the influence of the loop on the overall conformational stability of apoflavodoxin is quite low and the natively folded shortened variant (120–139) is almost as stable as the wild type protein. The fact that the loop, which is not required for a proper folding of the polypeptide, does not even play a significant role in increasing the conformational stability of the protein supports our proposal (36) that the differentiating loop of long-chain flavodoxins may be related to a recognition function, rather than serving a structural purpose.

Received for publication, May 25, 2004 , and in revised form, July 21, 2004.

^* This work has been supported in part by Grants BMC 2001-252, PB91-0368, BI095-2068, BIO2002-00720 and BQU/2000-1500, and BIO2000-1437 of MCYT from the Spanish Ministry of Science and Technology and FEDER funds, and by Grant P120/2001 from the Aragonese Government (Diputación General de Aragón). 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 Supplemental Equations 1, 2, 3, 4, 5, 6 and figure.

^¶ Supported by Bask Government fellowships.

^** Supported by the Spanish Ministry of Science and Technology.

Supported by a fellowship from the Aragonese Government (DGA).

^¶¶ Present address: Dept. of Biochemistry, University of Cambridge, Cambridge CB21GA, United Kingdom.

^|||| To whom correspondence should be addressed: Dept. Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain. E-mail: jsancho{at}unizar.es.

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