Evidence, from simulations, of a single state with residual native structure at the thermal denaturation midpoint of a small globular protein.

The folding of the B-domain of staphylococcal protein A has been studied by coarse-grained canonical and multiplexed replica-exchange molecular dynamics simulations with the UNRES force field in a broad range of temperatures (270 K < or = T < or = 350 K). In canonical simulations, the folding was found to occur either directly to the native state or through kinetic traps, mainly the topological mirror image of the native three-helix bundle. The latter folding scenario was observed more frequently at low temperatures. With increase of temperature, the frequency of the transitions between the folded and misfolded/unfolded states increased and the folded state became more diffuse with conformations exhibiting increased root-mean-square deviations from the experimental structure (from about 4 A at T = 300 K to 8.7 A at T = 325 K). An analysis of the equilibrium conformational ensemble determined from multiplexed replica exchange simulations at the folding-transition temperature (T(f) = 325 K) showed that the conformational ensemble at this temperature is a collection of conformations with residual secondary structures, which possess native or near-native clusters of nonpolar residues in place, and not a 50-50% mixture of fully folded and fully unfolded conformations. These findings contradict the quasi-chemical picture of two- or multistate protein folding, which assumes an equilibrium between the folded, unfolded, and intermediate states, with equilibrium shifting with temperature but with the native conformations remaining essentially unchanged. Our results also suggest that long-range hydrophobic contacts are the essential factor to keep the structure of a protein thermally stable.