How cooperative are protein folding and unfolding transitions?

Abstract

A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two-state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non-cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier-less "downhill" folding, as well as for continuous "uphill" unfolding transitions, indicate that gradual non-cooperative processes may be ubiquitous features on the free energy landscape of protein folding.

Keywords: cooperativity; downhill folding; intermediates; one-state; population distributions; uphill unfolding.

Figure 1

Experimental determination of cooperativity. A: Single exponential kinetics of the unfolding of monellin probed by fluorescence (upper panel; kinetic traces of unfolding in increasing denaturant concentration are shown from right to left) suggested a “two‐state” cooperative transition. B: Site‐specific unfolding/opening of four cysteine side chains (upper panel), probed by thiol labeling (SX) under native conditions, detected the presence of at least four discrete energy barriers.217 This, along with steady state and TR FRET measurements39, 60 indicated limited cooperativity in the unfolding transition of monellin. C: Increasing structural resolution, by using hydrogen exchange (HX) which probes the backbone hydrogen bonding network in multiple sequence segments of the protein (upper panel), revealed a multitude of small barriers, of the order of thermal energy, resulting in a gradual uphill unfolding transition.62, 81

Figure 2

Equilibrium unfolding transitions at single amino acid resolution. Urea‐induced unfolding transitions were measured for individual residues of bovine PrP_121–230 by NMR. A: The unfolding transition measured for each amino acid residue, shown on the protein structure in (B) was fit to a two‐state N↔U model. The observed dispersion in the thermodynamic parameters obtained from the fits, which reflect the residue‐specific sensitivity to urea denaturation, demonstrated the lack of thermodynamic cooperativity in the unfolding of the prion protein. Reprinted, with permission, from reference 66.

Figure 3

Gradual evolution of distance distributions in a kinetic unfolding experiment. The distributions of four distances in monellin, indicated on the protein structure in the top most panels, were monitored by multisite TR‐FRET, during unfolding in the presence of 4 M GdnHCl. Unimodal distributions for one of the distances (Cys68‐TNB) provided strong evidence of a continuous noncooperative expansion during unfolding. The bimodal distributions in the remaining three cases were centered at distances intermediate between those of the N and U states, suggesting the presence of intermediate ensembles. Continuous shifts in the distance distributions of the intermediate states provided further evidence for gradual noncooperative structural change. The data could be accounted for by a model in which structure was lost continuously during the gradual swelling of the protein during unfolding, which was describable by the Rouse model of polymer physics. Reproduced from Ref. 60.

Figure 4

Delineation of cooperativity from population distributions. Mass distributions measured by HX‐MS in the EX1 regime could directly differentiate between a cooperative and a gradual transition in monellin. Unimodal mass distributions at all time points of exchange, in 0 M GdnHCl (A and B) were indicative of a one‐state gradual transition under native conditions, involving the opening of one backbone amide site at a time. The solid vertical lines in each panel indicate the mass distributions corresponding to the N state (at 5 s), U state (at the final time point of exchange) and at the end of each kinetic phase of exchange. The observation of bimodality upon the addition of 1 M GdnHCl (C and D) indicated a switch to a cooperative transition, which involved the all‐or‐none opening of a subset of 14 backbone amide sites in the protein core, during the slow global unfolding step. Reprinted, with permission, from Ref. 62.

Figure 5

Tuning the cooperativity of protein folding/unfolding reactions. Changes in the stabilities of the N and U states, via changes in solvent conditions62 or mutations of the protein sequence, can switch a barrier‐limited cooperative process to a barrier‐less transition. The point of intersection of the N and U state energy wells corresponds to the position of the TS along the reaction coordinate. The resultant free energy landscape is shown in grey. Reprinted, with permission, from Ref. 62.

Figure 6

Ruggedness on the protein folding energy landscape. A subset of backbone amide hydrogens in monellin, which unfold gradually in native conditions (A), were induced to unfold cooperatively upon the addition of GdnHCl (B),62 suggesting that denaturant smoothens the roughness of the protein folding energy landscape. This was further confirmed by the observation that the unfolding kinetics of a sequence segment spanning two β strands in the protein, simplifies from being a triple exponential in native conditions to a single exponential reaction in the presence of GdnHCl (C).81 Denaturant smoothens the free energy landscape of protein folding (in gray), by reducing the intervening energetic barriers and kinetic traps present on a rough landscape (in brown). Reprinted, with permission, from Refs. 62 and 81.

Figure 7

Structural transitions in a gradual unfolding reaction. HX‐MS kinetics was measured for different sequence segments of monellin, generated by chemical fragmentation subsequent to exchange in 0M GdnHCl. Mapping the extents of exchange in individual sequence segments at different times of unfolding on to the protein structure revealed a diffuse and asynchronous dissolution of the backbone hydrogen bonding network in monellin. A dispersion in the extents of exchange within the α‐helix, demonstrated the noncooperativity in the unfolding of individual secondary structural elements in the protein. A lack of modular architecture and distinct secondary structural units in monellin, evident in the above representation, provided a structural basis for the gradual unfolding of the protein under native conditions. Reprinted, with permission, from Ref. 81.