title: Shifting transition states in the unfolding of a large ankyrin repeat protein.
abstract: The 33-amino-acid ankyrin motif comprises a beta-turn followed by two anti-parallel alpha-helices and a loop and tandem arrays of the motif pack in a linear fashion to produce elongated structures characterized by short-range interactions. In this article we use site-directed mutagenesis to investigate the kinetic unfolding mechanism of D34, a 426-residue, 12-ankyrin repeat fragment of the protein ankyrinR. The data are consistent with a model in which the N-terminal half of the protein unfolds first by unraveling progressively from the start of the polypeptide chain to form an intermediate; in the next step, the C-terminal half of the protein unfolds via two pathways whose transition states have either the early or the late C-terminal ankyrin repeats folded. We conclude that the two halves of the protein unfold by different mechanisms because the N-terminal moiety folds and unfolds in the context of a folded C-terminal moiety, which therefore acts as a "seed" and confers a unique directionality on the process, whereas the C-terminal moiety folds and unfolds in the context of an unfolded N-terminal moiety and therefore behaves like a single-domain ankyrin repeat protein, having a high degree of symmetry and consequently more than one unfolding pathway accessible to it.
authors.affiliation: MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ, United Kingdom.
authors.forename: Nicolas D
authors.initials: ND
authors.lastname: Werbeck
authors.forename: Pamela J E
authors.initials: PJ
authors.lastname: Rowling
authors.forename: Vasuki R
authors.initials: VR
authors.lastname: Chellamuthu
authors.forename: Laura S
authors.initials: LS
authors.lastname: Itzhaki
mesh_terms: D017089:Ankyrin Repeat / Q000235:genetics; D017487:Ankyrins / Q000737:chemistry* / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D002942:Circular Dichroism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D044367:Phase Transition; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
chemical_list: C110202:ANK1 protein, human; D017487:Ankyrins; D010446:Peptide Fragments; D011994:Recombinant Proteins