Abstract
One of the most intriguing predictions of energy landscape models is the existence of non-exponential protein folding kinetics caused by hierarchical structures in the landscapes. Here we provide the strongest evidence so far of such hierarchy and determine the time constants and weights of the kinetic components of the suggested hierarchic energy landscape. To our knowledge, the idea of hierarchical folding energy barriers has never been tested over such a broad timescale. Refolding of yeast phosphoglycerate kinase was initiated from the guanidine-unfolded state by stopped-flow or manual mixing and monitored by tryptophan fluorescence from 1 ms to 15 min. The strategy to build a model that describes folding of yeast phosphoglycerate kinase was to start from the simplest paradigm and modify it stepwise to the necessary minimal extent after repeated comparisons with the experiments. We made no a priori assumptions about the folding landscape. The result was a hierarchic finite level landscape model that quantitatively describes the refolding of yeast phosphoglycerate kinase from 1 ms to 15 min. The early steps of the folding process happen in the upper region of the landscape, where the surface has a hierarchic structure. This leads to stretched kinetics in the early phase of the folding. The lower region of the energy landscape is dominated by a trap that reflects the accumulation of molten globule intermediate state. From this intermediate, the protein can reach the global energy minimum corresponding to the native state through a cross-barrier folding step.
Highlights
One of the most intriguing predictions of energy landscape models is the existence of non-exponential protein folding kinetics caused by hierarchical structures in the landscapes
Stretched refolding kinetics has been observed experimentally and explained using the energy landscape concept (23, 26 –28). These findings provide experimental tests of hierarchic energy landscapes, and show examples of kinetics that stretch over several orders of magnitude in time, the models presented to explain the results are based on a priori assumptions about the shape of the energy landscape
Based on the above comparison of the refolding kinetics of the wild-type protein and the HisPGK P204H mutant, we can state that the transition from the unfolded into the intermediate state during the refolding of the Yeast phosphoglycerate kinase (yPGK) is directed by a scaled energy landscape
Summary
One of the most intriguing predictions of energy landscape models is the existence of non-exponential protein folding kinetics caused by hierarchical structures in the landscapes. 29, H-1113 Budapest, Hungary, the ¶Research Group for Biophysics, Hungarian Academy of Sciences-Semmelweis University, Puskin u. One of the most interesting predictions of landscape models is the existence of hierarchically structured protein folding energy surfaces that produce stretched folding kinetics that lack a characteristic time scale [25].
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