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J. Biol. Chem., Vol. 281, Issue 34, 24345-24350, August 25, 2006

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Tracking the Unfolding Pathway of a Multirepeat Protein via Tryptophan Scanning

EVIDENCE OF LOCALIZED INSTABILITY IN THE MITOCHONDRIAL IMPORT RECEPTOR Tom70^*

Simon R. Bushell¹, Stephen P. Bottomley², Jamie Rossjohn³, and Travis Beddoe⁴

From the Protein Crystallography Unit, Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics and the Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia

The tetratricopeptide repeat (TPR) is a degenerate 34-amino acid repeating motif that forms a repeating helix-turn-helix structure and is a well characterized mediator of protein-protein interactions. Recently, a biophysical investigation on one naturally occurring TPR protein, Tom70, found that the mitochondrial receptor displayed an unusual three-state unfolding pathway, distinct from the two-state model usually displayed by TPR proteins. To investigate this unusual behavior, we undertook a tryptophan-scanning analysis of Tom70, where both native and engineered tryptophan residues are used as fluorescent reporters to monitor the range of local and global unfolding events that comprise the unfolding pathway of Tom70. Specifically, seven Tom70 variants were constructed, each with a single tryptophan residue in each of the seven TPR repeats of Tom70. By combining equilibrium and kinetic fluorescent unfolding assays, with circular dichroism experiments, our study reveals that the unusual folding pathway of Tom70 is a consequence of the unfolding of two separate, autonomous TPR arrays, with the less stable region appearing to account for the low structural stability of Tom70.

Received for publication, March 29, 2006 , and in revised form, May 19, 2006.

^* This work was supported by the Australian Research Council. 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 an Australian Postgraduate Award Scholarship.

² A RD Wright Research Fellow and a Senior Logan Fellow.

³ Supported by an Australian Research Council Professorial Fellowship. To whom correspondence may be addressed. Tel.: 61-3-9905-3736; Fax: 61-3-9905-4699; E-mail: Jamie.Rossjohn{at}med.monash.edu.au. ⁴Supported by a Peter Doherty Fellowship from the National Health and Medical Research Council. To whom correspondence may be addressed. Tel.: 61-3-9905-3736; Fax: 61-3-9905-4699; E-mail: Travis.Beddoe{at}med.monash.edu.au.

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