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快乐十分前三组推荐号:Fast pressure-jump all-atom simulations and experiments reveal site-specific protein dehydration-folding dynamics
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When proteins fold, water is squeezed out of the hydrophobic core, leaving small desolvated pockets. Such voids are critical for protein flexibility and function, but not much is known about how and when they form during protein folding. We combine long atomic-level simulations with rapid pressure-drop experiments to see how water gets out of a protein as it folds. For one small protein, we find that dry pockets can form repeatedly between various pairs of protein α-helices before a successful folding event finally occurs. For another small protein, drying and folding are much more concerted. Thus, the formation of void pockets that enhance protein flexibility can occur in a concerted fashion or by coincidence of several local events.
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 (λ6–85) 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 nonperturbative 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 (α3D)], whose drying and core formation proceed in concert, λ6–85 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.
?1M.B.P. and Y.Z. contributed equally to this work.
?2Present address: Department of Physics, Stanford University, Stanford, CA 94305.
- ?3To whom correspondence may be addressed. Email: or .
Author contributions: K.S., M.G., and T.V.P. designed research; M.B.P., Y.Z., and T.V.P. performed research; M.B.P., Y.Z., K.S., M.G., and T.V.P. analyzed data; and M.B.P., Y.Z., M.G., and T.V.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1814927116/-/DCSupplemental.
Published under the PNAS license.