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J. Biol. Chem., Vol. 279, Issue 36, 37261-37270, September 3, 2004

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Role of Kinetic Intermediates in the Folding of Leech Carboxypeptidase Inhibitor^*

Joan L. Arolas, Sílvia Bronsoms, Julia Lorenzo, Francesc X. Aviles¶, Jui-Yoa Chang||, and Salvador Ventura**

From the Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain and the ^||Research Center for Protein Chemistry, Institute of Molecular Medicine and Department of Biochemistry and Molecular Biology, The University of Texas, Houston, Texas 77030

The oxidative folding and reductive unfolding pathways of leech carboxypeptidase inhibitor (LCI; four disulfides) have been characterized in this work by structural and kinetic analysis of the acid-trapped folding intermediates. The oxidative folding of reduced and denatured LCI proceeds rapidly through a sequential flow of 1-, 2-, 3-, and 4-disulfide (scrambled) species to reach the native form. Folding intermediates of LCI comprise two predominant 3-disulfide species (designated as III-A and III-B) and a heterogeneous population of scrambled isomers that consecutively accumulate along the folding reaction. Our study reveals that forms III-A and III-B exclusively contain native disulfide bonds and correspond to stable and partially structured species that interconvert, reaching an equilibrium prior to the formation of the scrambled isomers. Given that these intermediates act as kinetic traps during the oxidative folding, their accumulation is prevented when they are destabilized, thus leading to a significant acceleration of the folding kinetics. III-A and III-B forms appear to have both native disulfides bonds and free thiols similarly protected from the solvent; major structural rearrangements through the formation of scrambled isomers are required to render native LCI. The reductive unfolding pathway of LCI undergoes an apparent all-or-none mechanism, although low amounts of intermediates III-A and III-B can be detected, suggesting differences in protection against reduction among the disulfide bonds. The characterization of III-A and III-B forms shows that the former intermediate structurally and functionally resembles native LCI, whereas the III-B form bears more resemblance to scrambled isomers.

Received for publication, May 19, 2004 , and in revised form, June 24, 2004.

^* This work was supported by Grant BIO2001-2046 (Ministerio de Ciencia y Tecnología, MCYT, Spain) and by the Centre de Referència en Biotecnologia (Generalitat de Catalunya, 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.

Recipient of a fellowship from the Universitat Autònoma de Barcelona.

^¶ To whom correspondence may be addressed. Tel.: 34-93-581-1315; Fax: 34-93-581-2011; E-mail: fxaviles{at}einstein.uab.es. ** Supported by a Ramón y Cajal project awarded by the MCYT and co-financed by the Universitat Autònoma de Barcelona. To whom correspondence may be addressed. Tel.: 34-93-581-1315; Fax: 34-93-581-2011; E-mail: salvador.ventura{at}uab.es.

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