Criteria for downhill protein folding: calorimetry, chevron plot, kinetic relaxation, and single-molecule radius of gyration in chain models with subdued degrees of cooperativity.

Recent investigations of possible downhill folding of small proteins such as BBL have focused on the thermodynamics of non-two-state, "barrierless" folding/denaturation transitions. Downhill folding is noncooperative and thermodynamically "one-state," a phenomenon underpinned by a unimodal conformational distribution over chain properties such as enthalpy, hydrophobic exposure, and conformational dimension. In contrast, corresponding distributions for cooperative two-state folding are bimodal with well-separated population peaks. Using simplified atomic modeling of a three-helix bundle-in a scheme that accounts for hydrophobic interactions and hydrogen bonding-and coarse-grained C(alpha) models of four real proteins with various degrees of cooperativity, we evaluate the effectiveness of several observables at defining the underlying distribution. Bimodal distributions generally lead to sharper transitions, with a higher heat capacity peak at the transition midpoint, compared with unimodal distributions. However, the observation of a sigmoidal transition is not a reliable criterion for two-state behavior, and the heat capacity baselines, used to determine the van't Hoff and calorimetric enthalpies of the transition, can introduce ambiguity. Interestingly we find that, if the distribution of the single-molecule radius of gyration were available, it would permit discrimination between unimodal and bimodal underlying distributions. We investigate kinetic implications of thermodynamic noncooperativity using Langevin dynamics. Despite substantial chevron rollovers, the relaxation of the models considered is essentially single-exponential over an extended range of native stabilities. Consistent with experiments, significant deviations from single-exponential behavior occur only under strongly folding conditions.