What have we learned from the studies of two-state folders, and what are the unanswered questions about two-state protein folding?

Abstract

Small proteins with globular structures often fold by simple all-or-none mechanisms, both in an equilibrium and a kinetic sense, despite the very large number of partly folded conformations available. This type of 'two-state' folding will be discussed in terms of experimental tests, underlying molecular mechanisms, and limits to two-state behavior. Factors that appear to be important for two-state folding include topology (sequence distance of contacts in the native structure), molecular cooperativity and local energy distribution. Because their local stability distributions and cooperativities can be dissected and analyzed separately from topological features, recent studies of the folding of symmetric proteins will be discussed as a means to better understand the origins of two-state folding.

Figure 1. Equilibrium two-state folding

(A) On a single-molecule basis, an observable parameter that is sensitive to structure (such as intrinsic fluorescence or far-UV CD) takes on only two values corresponding to the native and denatured state. The average of the time spent at each value reflects the equilibrium constant for folding. For a kinetically two-state mechanism, the transitions are expected to be abrupt in time, as shown. (B) In an ensemble average, a steep but continuous transition is seen between native and denatured baselines (dashed and dotted lines, respectively). Although the average equilibrium signal lies at an intermediate value between these two limits, individual members of the ensemble remain restricted to the native and denatured states (as depicted in panel A). Data are for CD-monitored unfolding of the Notch ankyrin domain, reproduced from (Street et al., 2008) with permission.

Figure 2. Kinetic two-state folding

(A) A highly simplified reaction diagram for two-state protein folding, in which Gibbs free energy is depicted as a function of a general reaction coordinate ξ, which resolves the native, denatured, and transition state ensembles. In terms of a free energy diagram, a single transition-state ensemble (indicated by the double-dagger) acts as a barrier to folding, determining the rate constant for folding and unfolding. (B) A linear chevron plot for a kinetically two-state protein. The solid line depicts k_app; k_f and k_u are shown as dotted and dashed lines, respectively. Linear folding limbs are obtained if the transition state depicted in panel (A) remains the highest free energy point on the dominant routes to folding (i.e. no new barriers appear (Sanchez & Kiefhaber, 2003)), and does not change its structure with denaturant (Otzen et al., 1999).