Diffusing and colliding: the atomic level folding/unfolding pathway of a small helical protein

Proteins with ultra-fast folding/unfolding kinetics are excellent candidates for study by molecular dynamics. Here, we describe such simulations of a three helix bundle protein, the engrailed homeodomain (En-HD), which folds via the diffusion-collision model. The unfolding pathway of En-HD was characterized by seven simulations of the protein and 12 simulations of its helical fragments yielding over 1.1 micros of simulation time in water. Various conformational states along the unfolding pathway were identified. There is the compact native-like transition state, a U-shaped helical intermediate and an unfolded state with dynamic helical segments. Each of these states is in good agreement with experimental data. Examining these states as well as the transitions between them, we find the role of long-range tertiary contacts, specifically salt-bridges, important in the folding/unfolding pathway. In the folding direction, charged residues form long-range tertiary contacts before the hydrophobic core is formed. The formation of HII is assisted by a specific salt-bridge and by non-specific (fluctuating) tertiary contacts, which we call contact-assisted helix formation. Salt-bridges persist as the protein approaches the transition state, stabilizing HII until the hydrophobic core is formed. To complement this information, simulations of fragments of En-HD illustrate the helical propensities of the individual segments. By thermal denaturation, HII proved to be the least stable helix, unfolding in less than 450 ps at high temperature. We observed the low helical propensity of C-terminal residues from HIII in fragment simulations which, when compared to En-HD unfolding simulations, link the unraveling of HIII to the initial event that drives the unfolding of En-HD.