Microsecond folding dynamics of the F13W G29A mutant of the B domain of staphylococcal protein A by laser-induced temperature jump

107

Efficient protein folding implies a microscopic funnel-like multidimensional free-energy landscape. Macroscopically, conformational entropy reduction can manifest itself as part of an empirical barrier in the traditional view of folding, but experiments show that such barriers can also entail significant unfavorable enthalpy changes. This observation raises the puzzling possibility, irrespective of conformational entropy, that individual microscopic folding trajectories may encounter large uphill moves and thus the multidimensional free-energy landscape may not be funnel-like. Here, we investigate how nanoscale hydrophobic interactions might underpin this salient enthalpic effect in biomolecular assembly by computer simulations of the association of two preformed polyalanine or polyleucine helices in water. We observe a high, positive enthalpic signature at room temperature when the helix separation is less than a single layer of water molecules. Remarkably, this unfavorable enthalpy change, with a parallel increase in void volume, is largely compensated for by a concomitant increase in solvent entropy, netting only a small or nonexistent microscopic free-energy barrier. Thus, our findings suggest that high enthalpic folding barriers can be consistent with a funnel picture of folding and are mainly a desolvation phenomenon indicative of a cooperative mechanism of simultaneous formation of multiple side-chain contacts at the rate-limiting step.

Section: Ramifications For the Transition States Of Protein Foldingmentioning

confidence: 65%

mentioning

confidence: 94%

Hydrophobic association of α-helices, steric dewetting, and enthalpic barriers to protein folding

MacCallum

Moghaddam

Chan

et al. 2007

107

“…Other ultra-fast folding proteins have been analyzed successfully in terms of a two-state process (22,23), implying that barrier crossing reaction is an appropriate description for these systems as well. Generally, proteins fold cooperatively, either at the global, domain, or subglobal level (24)(25)(26).…”

Section: Resultsmentioning

confidence: 99%

Barrier-limited, microsecond folding of a stable protein measured with hydrogen exchange: Implications for downhill folding

Meisner

Sosnick

2004

Folding experiments are conducted to test whether a covalently cross-linked coiled-coil folds so quickly that the process is no longer limited by a free-energy barrier. This protein is very stable and topologically simple, needing merely to ''zipper up,'' while having an extrapolated folding rate of kf ‫؍‬ 2 ؋ 10 5 s ؊1 . These properties make it likely to attain the elusive ''downhill folding'' limit, at which a series of intermediates can be characterized. To measure the ultra-fast kinetics in the absence of denaturant, we apply NMR and hydrogen-exchange methods. protein folding ͉ EX1 ͉ stretched exponential ͉ coiled-coil T he two-state approximation of protein folding, in which the unfolded and native states are separated by a single freeenergy barrier and no other species accumulate to a significant degree, appears to be adequate for most small proteins (1). Nevertheless, it is unsatisfying because it generally precludes identifying the individual steps involved in the folding process. To overcome this problem, experiments (2-6) have pursued the elusive theoretical prediction of ''downhill folding'' (7-9) (Fig. 1). If attainable, a downhill energy surface may allow the experimental identification of a series of intermediate states along the folding pathways that likely exists but largely has been observed only in computer simulations.Downhill-folding behavior is predicted to occur when folding rates approach the value of the attempt frequency of the barrier crossing process,Here, the barrier height does not contribute to the rate, and ⌬G ‡ Ϸ0. This limit may be likened to extreme Hammond behavior (10) in which the folding transition state moves so far to the starting condition in response to heightened stability that the transition state coincides with the unfolded state.A barrier-free reaction must be extremely rapid, but how fast is fast enough? Estimates from measured rates of helix, loop, and hairpin formation, reaction-rate and polymer-collapse theories, and folding simulations suggest that the minimum possible folding time constant is on the order of 1 s (6). Because of the increased ruggedness of the energy landscape, downhill folders of high stability would have proportionally slower folding rates, as would those with longer sequences and greater ␤-sheet content. After correcting for length and stability, 12 proteins with predicted barrier-free time constants of Ͻ100 s have been identified as potential downhill folders (6).The folding speed and stability of the covalently cross-linked variant of the dimeric yeast transcription factor GCN4 coiledcoil places it with the top members of this group (11,12). For the version GCN4p2C, with the Gly-Gly-Cys tether located at the C terminus, the extrapolated folding time constant is f Ϸ 10 s and Ͻ1 s when normalized for the high stability of the molecule (6). This fast folding rate may be due to a simple topology that requires that the helices only ''zipper-up.'' These qualities identify it among the most likely candidates for downhill folding. Here, we invest...

“…Single-molecule experiments can give conclusive answers. The B domain of Protein A (BDPA) is an ultrafast folding protein (17,36,37). Its refolding rate constant at 298 K in water is 10 5 s Ϫ1 (24).…”

Section: Resultsmentioning

confidence: 99%

Distinguishing between cooperative and unimodal downhill protein folding

Huang

Sato²,

Sharpe³

et al. 2007

100

136

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...