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Journal Article

Shifting transition states in the unfolding of a large ankyrin repeat protein


Werbeck,  Nicolas D.
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;

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Werbeck, N. D., Rowling, P. J. E., Chellamuthu, V. R., & Itzhaki, L. S. (2008). Shifting transition states in the unfolding of a large ankyrin repeat protein. Proceedings of the National Academy of Sciences of the United States of America (Astor), 105(29), 9982-9987. doi:10.1073/pnas.0705300105.

Cite as: http://hdl.handle.net/11858/00-001M-0000-002C-A64E-1
The 33-amino-acid ankyrin motif comprises a β-turn followed by two anti-parallel α-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.