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 lambda-repressor fragment (lambda(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 alpha-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 alpha-helices 1 and 3 is reflected in the rate-limiting folding step, fast pressuredrop 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-alpha-helix bundle (alpha 3D)], whose drying and core formation proceed in concert, lambda(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.