Multiple protein folding nuclei and the transition state ensemble in two-state proteins

Using exhaustive simulations of lattice models with side-chains, we show th at optimized two-state folders reach the native state by a nucleation-colla pse mechanism with multiple folding nuclei (MFN). For both the full model a nd the Go version, there are certain contacts that on an average participat e in the critical nuclei with higher probability than the others. The high- (greater than or similar to0.5) probability contacts are largely determine d by the structure of the native state. Comparison of the results for the f ull sequence and the Go model shows that non-native interactions compromise the degree of cooperativity and stability of the native state. From an ext remely detailed analysis of the folding kinetics, we find that non-native i nteractions are present in the folding nuclei. The folding times decrease i f the non-native interactions in the folding nuclei are made neutral or rep ulsive. Using cluster analysis and making no prior assumption about reactio n coordinate, we show that both full and Go models have three distinct tran sition states that give a structural description for the MFN, In the transi tion states, on an average, about two-thirds of the sequence is structured, whereas the rest is disordered, reminiscent of the polarized transition st ate in the SH3 domain. Our studies show that Go models cannot describe the transition state characteristics of two-state folders at the molecular leve l. As a byproduct of our investigations, we establish that our method of co mputing the transition state ensemble is numerically equivalent to the tech nique based on the stochastic separatrix, which also does not require a pri ori knowledge of the folding reaction coordinate. Proteins 2001;43:465-475. (C) 2001 Wiley-Liss, Inc.