title: Tracking the unfolding pathway of a multirepeat protein via tryptophan scanning: evidence of localized instability in the mitochondrial import receptor Tom70.
abstract: 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.
authors.affiliation: Protein Crystallography Unit, Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Clayton, Victoria 3800, Australia.
authors.forename: Simon R
authors.initials: SR
authors.lastname: Bushell
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Bottomley
authors.forename: Jamie
authors.initials: J
authors.lastname: Rossjohn
authors.forename: Travis
authors.initials: T
authors.lastname: Beddoe
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D024101:Mitochondrial Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D017510:Protein Folding; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D014364:Tryptophan
chemical_list: D024101:Mitochondrial Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D014364:Tryptophan