Intermediates: ubiquitous species on folding energy landscapes?

Abstract

Although intermediates have long been recognised as fascinating species that form during the folding of large proteins, the role that intermediates play in the folding of small, single-domain proteins has been widely debated. Recent discoveries using new, sensitive methods of detection and studies combining simulation and experiment have now converged on a common vision for folding, involving intermediates as ubiquitous stepping stones en route to the native state. The results suggest that the folding energy landscapes of even the smallest proteins possess significant ruggedness in which intermediates stabilized by both native and non-native interactions are common features.

Figure 1

Example chevron plots (top) and free energy diagrams (bottom) for different folding scenarios. Each chevron plot shows the denaturant ([Den]) dependence of the observed rate constant (ln k). (a) Barrier-less folding. (b) Classical two-state folding, in which intermediates are not populated. (c) Folding via a high-energy intermediate. The switch between early and late transition states as a function of denaturant concentration results in non-linearity of the folding and unfolding branches of the chevron plot. (d) Population of a late folding intermediate, subsequent to the rate-limiting transition state. Such proteins might display two-state kinetics and the population of intermediates is typically inferred from native state hydrogen exchange experiments. (e) Folding via the population of a stable intermediate in the first millisecond of folding. This is revealed by the so-called ‘rollover’ in the folding branch of the chevron plot determined using stopped-flow methods (closed circles). In the case where the intermediate accumulates rapidly during folding, a second chevron plot reflecting the formation and unfolding of the intermediate can be obtained using ultra-rapid mixing experiments (open circles). The positions of the denatured state (U), intermediate state (I), early and late transition states (TS1 and TS2, respectively) and native state (N) are shown on an arbitrary scale.