Distinguishing between cooperative and unimodal downhill protein folding

Conventional cooperative protein folding invokes discrete ensembles of native and denatured state structures in separate free-energy wells. Unimodal noncooperative (`downhill`) folding, however, proposes an ensemble of states occupying a single free-energy well for proteins folding at >/=4 x 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 time-averaged 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 x 10(4) s(-1) in denaturant solution, extrapolating to 2 x 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.