Distinguishing between cooperative and unimodal downhill protein folding

Abstract

Conventional cooperative protein folding invokes discrete ensembles of native and denatured state structures in separate freeenergy wells. Unimodal noncooperative (''downhill'') folding, however, proposes an ensemble of states occupying a single free-energy well for proteins folding at >4 ؋ 10 4 s ؊1 at 298 K. It is difficult to falsify unimodal mechanisms for such fast folding proteins by standard equilibrium experiments because both cooperative and unimodal mechanisms can present the same timeaveraged structural, spectroscopic, and thermodynamic properties when the time scale used for observation is longer than for equilibration. However, kinetics can provide the necessary evidence. Chevron plots with strongly sloping linear refolding arms are very difficult to explain by downhill folding and are a signature for cooperative folding via a transition state ensemble. The folding kinetics of the peripheral subunit binding domain POB and its mutants fit to strongly sloping chevrons at observed rate constants of >6 ؋ 10 4 s ؊1 in denaturant solution, extrapolating to 2 ؋ 10 5 s ؊1 in water. Protein A, which folds at 10 5 s ؊1 at 298 K, also has a well-defined chevron. Single-molecule fluorescence energy transfer experiments on labeled Protein A in the presence of denaturant demonstrated directly bimodal distributions of native and denatured states.A currently controversial subject in protein folding is unimodal ''downhill'' versus classical cooperative folding. It is generally accepted that proteins fold on a free-energy landscape in which there are ensembles of states separated by free-energy barriers (1). Accordingly, there are cooperative transitions between those ensembles of states, as, for example, the native N and denatured D ensembles, which have a bimodal distribution of properties. However, when there is an extreme energetic bias toward the native state, the protein may fold ''downhill,'' without an energy barrier (1). This conventional (''chemical'') view of folding has been challenged by Muñoz and colleagues (2, 3), who claim that for a protein NapBBL, a truncated and naphthylalanine-labeled derivative of the BBL peripheral subunit binding domain (PSBD) from Escherichia coli, in particular, and for all proteins that fold faster than 40,000 s Ϫ1 at 298 K (4), the D and N states are not separated by an energy barrier, but slowly merge into each other with changing conditions; Muñoz and colleagues (2) call this mechanism ''downhill'' folding ( Fig. 1). We use the term noncooperative or unimodal (5) for this downhill folding. Various criteria derived from equilibrium experiments have been proposed to be signatures of unimodal folding (2, 6). Downhill folding is proposed to be important, in particular as a means for the PSBDs to adjust their sizes as ''molecular rheostats,'' and in general because it is suggested (2, 4, 7) that it opens up the exciting prospect of examining the whole pathway of folding from equilibrium spectroscopic observations.Until recently, the proteins that were studied folded slowly com...

Direct observation of barrier-limited folding of BBL by single-molecule fluorescence resonance energy transfer

“…The key protein in the unimodal hypothesis is BBL, a member of the peripheral subunit binding domain family, which is claimed to be a global downhill folder based especially on the dispersion of melting temperature for individual residues (2). It is extremely difficult to falsify unimodal folding because most equilibrium and kinetic data can be interpreted by numerous alternative mechanisms, especially when the rate of interconversion of D and N states is on a faster time scale than that of observation (6). The dependence of rates of folding and unfolding on denaturant concentration is good evidence for the nature of the mechanism-classical chevron plots with a steep limb are compatible with only a folding scenario with a free energy barrier and a transition state, so there is a significant change of solvent-accessible surface between the ground and transition states (6)(7)(8).…”

“…That evidence combined with a rationalization of the anomalous behavior of BBL has been used as evidence that the folding of BBL can be accommodated by a barrier limited folding model, with some heterogeneity of the native state (9)(10)(11)(12). Direct observation of distinct states of BBL in the transition region at equilibrium would provide compelling evidence for barrier limited folding (6,13). The current method of choice is single-molecule fluorescence resonance energy transfer (SM-FRET) experiments, which has been widely applied in the studies of protein folding, structure, and function, and is especially useful in detection of heterogeneity of populations (14)(15)(16)(17)(18)(19)(20)(21).…”

“…We were not able to use SM-FRET to resolve unambiguously the folding mechanism of BBL because it folds in a fraction of a millisecond at room temperature, whereas the typical time resolution of SM-FRET was, until very recently, typically 1 ms (6,21,22), in which time BBL would rapidly equilibrate and appear as a time-averaged structure of the separate states if it folds by a barrier limited transition (23, 24). Here we study the folding of the protein by using improved equipment that allows study in the appropriate sub-millisecond time range.…”

“…It is extremely difficult to falsify unimodal folding because most equilibrium and kinetic data can be interpreted by numerous alternative mechanisms, especially when the rate of interconversion of D and N states is on a faster time scale than that of observation (6). The dependence of rates of folding and unfolding on denaturant concentration is good evidence for the nature of the mechanism-classical chevron plots with a steep limb are compatible with only a folding scenario with a free energy barrier and a transition state, so there is a significant change of solvent-accessible surface between the ground and transition states (6)(7)(8). That evidence combined with a rationalization of the anomalous behavior of BBL has been used as evidence that the folding of BBL can be accommodated by a barrier limited folding model, with some heterogeneity of the native state (9)(10)(11)(12).…”

“…We have applied SM-FRET to the B-domain of Protein A (BDPA) and directly observed the denatured and native states of this protein in its transition region (6), the signature of barrier-limited folding. We were not able to use SM-FRET to resolve unambiguously the folding mechanism of BBL because it folds in a fraction of a millisecond at room temperature, whereas the typical time resolution of SM-FRET was, until very recently, typically 1 ms (6,21,22), in which time BBL would rapidly equilibrate and appear as a time-averaged structure of the separate states if it folds by a barrier limited transition (23,24).…”