Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 105, Issue 29, Pages 9982-9987Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0705300105
Keywords
parallel pathways; protein engineering; protein folding; D34
Categories
Funding
- Medical Research Council [MC_U105370180] Funding Source: Medline
- MRC [MC_U105370180] Funding Source: UKRI
- Medical Research Council [MC_U105370180] Funding Source: researchfish
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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.
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