Experimental evidence for a frustrated energy landscape in a three-helix-bundle protein family

Abstract

Energy landscape theory is a powerful tool for understanding the structure and dynamics of complex molecular systems, in particular biological macromolecules1. The primary sequence of a protein defines its free energy landscape, and thus determines the folding pathway and the rate constants of folding and unfolding, as well as its native structure. Theory has shown that roughness in the energy landscape will lead to slower folding1, but derivation of detailed experimental descriptions of this landscape is challenging. Simple folding models2,3 show that folding is significantly influenced by chain entropy; proteins where the contacts are local fold fast, due to the low entropy cost of forming stabilising, native contacts during folding4,5. For some protein families, stability is also a determinant of folding rate constants6. Where these simple metrics fail to predict folding behaviour it is probable that there are features in the energy landscape that are unusual. Such general observations cannot explain the folding behaviour of the R15, R16 and R17 domains of α-spectrin. R15 folds ~3000 times faster than its homologues, although they have similar structures, stabilities and, as far as can be determined, transition state stabilities7-10. Here we show that landscape roughness (internal friction) is responsible for the slower folding and unfolding of R16 and R17. We use chimeric domains to demonstrate that this internal friction is a property of the core, and suggest that frustration in the landscape of the slow folding spectrin domains may be due to mis-docking of the long helices during folding. Although theoretical studies have suggested that rugged landscapes will result in slower folding, this is the first time that such a phenomenon has been shown experimentally to directly influence the folding kinetics of a “normal” protein with a significant energy barrier – one which folds on a relatively slow ms-s timescale.