Structured Pathway across the Transition State for Peptide Folding Revealed by Molecular Dynamics Simulations

Small globular proteins and peptides commonly exhibit two-state folding kinetics in which the rate limiting step of folding is the surmounting of a single free energy barrier at the transition state (TS) separating the folded and the unfolded states. An intriguing question is whether the polypeptide chain reaches, and leaves, the TS by completely random fluctuations, or whether there is a directed, stepwise process. Here, the folding TS of a 15-residue β-hairpin peptide, Peptide 1, is characterized using independent 2.5 μs-long unbiased atomistic molecular dynamics (MD) simulations (a total of 15 μs). The trajectories were started from fully unfolded structures. Multiple (spontaneous) folding events to the NMR-derived conformation are observed, allowing both structural and dynamical characterization of the folding TS. A common loop-like topology is observed in all the TS structures with native end-to-end and turn contacts, while the central segments of the strands are not in contact. Non-native sidechain contacts are present in the TS between the only tryptophan (W11) and the turn region (P7-G9). Prior to the TS the turn is found to be already locked by the W11 sidechain, while the ends are apart. Once the ends have also come into contact, the TS is reached. Finally, along the reactive folding paths the cooperative loss of the W11 non-native contacts and the formation of the central inter-strand native contacts lead to the peptide rapidly proceeding from the TS to the native state. The present results indicate a directed stepwise process to folding the peptide. Author Summary: The folding dynamics of many small protein/peptides investigated recently are in terms of simple two-state model in which only two populations exist (folded and unfolded), separated by a single free energy barrier with only one kinetically important transition state (TS). However, dynamical characterization of the folding TS is challenging. We have used independent unbiased atomistic molecular dynamics simulations with clear folding-unfolding transitions to characterize structural and dynamical features of transition state ensemble of Peptide 1. A common loop-like topology is observed in all TS structures extracted from multiple simulations. The trajectories were used to examine the mechanism by which the TS is reached and subsequent events in folding pathways. The folding TS is reached and crossed in a directed stagewise process rather than through random fluctuations. Specific structures are formed before, during, and after the transition state, indicating a clear structured folding pathway.