PREDICTING COUPLING LIMITS AND FOLDING KINETICS FROM AN EXPERIMENTALLY-DETERMINED FOLDING ENERGY LANDSCAPE

The Notch ankyrin domain is a repeat protein whose folding has been characterized through equilibrium and kinetic measurements. In previous work, equilibrium folding free energies of truncated Notch ankyrin domain constructs were used to generate an experimentally-determined folding energy landscape. Here, this folding energy landscape is used to investigate folding cooperativity and kinetics associated with the Notch ankyrin domain. To investigate the cooperative limits associated with the Notch ankyrin domain, we have generated 15 variants with single and multiple destabilizing substitutions that make the energy landscape uneven. By applying a free energy additivity analysis to these variants, we quantified the destabilization threshold over which repeats 6-7 decouple from repeats 1-5. The free energy coupling limit suggested by this additivity analysis (~4 kcal/mol) is also reflected in m-value analysis, and in differences between equilibrium unfolding transitions as monitored by circular dichroism versus fluorescence. All of these observations are quantitatively predicted by analyzing the response of the experimentally-determined energy landscape to increasing unevenness. These results highlight the importance of a uniform distribution of local stability in achieving cooperative unfolding. To investigate the folding kinetics of the Notch ankyrin domain, we parameterized a kinetic model in which local transition probabilities between partly folded states are based on energy values from the landscape. The landscape-based model correctly predicts highly diverse experimentally-determined kinetic features associated with folding of the Notch ankyrin domain and sequence variants. These predictions include single exponential folding and double exponential unfolding, curvature in the unfolding limb of the chevron plot, population of a transient unfolding intermediate, relative folding rates of variants over three orders of magnitude, and a change in the folding pathway that results from C-terminal stabilization. These findings indicate that the folding of the Notch ankyrin domain is thermodynamically controlled at a local level and demonstrate that accurate prediction of folding rates and pathways require both the complexity of an energy landscape and the quantitative determination of the energies on the landscape