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

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 i