Fast pressure-jump all-atom simulations and experiments reveal site-specific protein dehydration-folding dynamics

As theory and experiment have shown, protein dehydration is a major contributor to protein folding. Dehydration upon folding can be characterized directly by all-atom simulations of fast pressure drops, which create desolvated pockets inside the nascent hydrophobic core. Here, we study pressure-drop refolding of three λ-repressor fragment (λ ₆ – ₈₅ ) mutants computationally and experimentally. The three mutants report on tertiary structure formation via different fluorescent helix–helix contact pairs. All-atom simulations of pressure drops capture refolding and unfolding of all three mutants by a similar mechanism, thus validating the non-perturbative nature of the fluorescent contact probes. Analysis of simulated interprobe distances shows that the α-helix 1–3 pair distance displays a slower characteristic time scale than the 1–2 or 3–2 pair distance. To see whether slow packing of α-helices 1 and 3 is reflected in the rate-limiting folding step, fast pressure-drop relaxation experiments captured refolding on a millisecond time scale. These experiments reveal that refolding monitored by 1–3 contact formation indeed is much slower than when monitored by 1–2 or 3–2 contact formation. Unlike the case of the two-state folder [three–α-helix bundle (α ₃ D)], whose drying and core formation proceed in concert, λ ₆ – ₈₅ repeatedly dries and rewets different local tertiary contacts before finally forming a solvent-excluded core, explaining the non–two-state behavior observed during refolding in molecular dynamics simulations. This work demonstrates that proteins can explore desolvated pockets and dry globular states numerous times before reaching the native conformation.