Stability, cooperativity and folding kinetics on the Notch ankyrin domain.

摘要

Ankyrin repeat proteins have an elongated, periodic topology comprised exclusively of short-range interactions. Despite this modular architecture, the ankyrin domain from the Drosophila Notch receptor undergoes two-state unfolding. To determine the limits of this cooperativity, and the effect of structural modularity on the folding mechanism, we characterized the stability and folding kinetics of the Notch ankyrin domain and a series of variants. The substitutions are of analogous alanines to glycine in each repeat, and allow the same perturbation to be examined at different positions in the protein.; Whereas cooperativity is maintained in the presence of destabilizing substitutions in repeats two through five, the analogous substitutions in repeats six and seven disrupt cooperativity. The effect of multiple perturbations on stability suggests that destabilization of repeat seven disrupts repeat six without affecting repeats one through five. These effects are consistent with an asymmetric distribution of stability in which unstable C-terminal repeats are connected to stable N-terminal repeats by a stabilizing interface.; Refolding of the Notch ankyrin domain is six orders of magnitude slower than that predicted from its local topology. The major observed folding phase involves a single barrier that is not limited by prolyl isomerization or the formation of an intermediate. To determine if structural redundancy in the native state leads to multiple parallel pathways for folding, we measured the effect of the destabilizing substitutions on folding kinetics. The strong effect of substitutions in a subset of repeats is consistent with a discrete kinetic pathway that can be slowed by specific destabilizations. Φ-value analysis reveals that the environment surrounding the alanines in repeats three through five has a native-like structure in the transition state ensemble, whereas the analogous environment in repeats two, six and seven is disordered. The formation of early structure in repeats three through five suggests the importance of both stabilizing repeat interfaces and residual structure in the denatured state in dictating a folding mechanism. The slow folding rate may be due to the potential for forming non-native interfaces that must be disrupted before the native state is formed, and to the low intrinsic stability of the individual repeats.