title: Misplaced helix slows down ultrafast pressure-jump protein folding.
abstract: Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein λ*YA, the Y22W/Q33Y/G46,48A mutant of λ-repressor fragment 6-85, from 3 μs to 5 ms after a 1,200-bar P-drop. In addition to a microsecond phase, we observe a slower 1.4-ms phase during refolding to the native state. Unlike temperature denaturation, pressure denaturation produces a highly reversible helix-coil-rich state. This difference highlights the importance of the denatured initial condition in folding experiments and leads us to assign a compact nonnative helical trap as the reason for slower P-jump-induced refolding. To complement the experiments, we performed over 50 μs of all-atom molecular dynamics P-drop refolding simulations with four different force fields. Two of the force fields yield compact nonnative states with misplaced α-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of λ*YA contains mainly residual helix and little β-sheet; following a fast P-drop, at least some λ*YA forms misplaced helical structure within microseconds. We hypothesize that nonnative helix at helix-turn interfaces traps the protein in compact nonnative conformations. These traps delay the folding of at least some of the population for 1.4 ms en route to the native state. Based on molecular dynamics, we predict specific mutations at the helix-turn interfaces that should speed up refolding from the pressure-denatured state, if this hypothesis is correct.
authors.affiliation: Department of Chemistry, Center for the Physics of Living Cells and Beckman Institute, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Maxim B
authors.initials: MB
authors.lastname: Prigozhin
authors.forename: Yanxin
authors.initials: Y
authors.lastname: Liu
authors.forename: Anna Jean
authors.initials: AJ
authors.lastname: Wirth
authors.forename: Shobhna
authors.initials: S
authors.lastname: Kapoor
authors.forename: Roland
authors.initials: R
authors.lastname: Winter
authors.forename: Klaus
authors.initials: K
authors.lastname: Schulten
authors.forename: Martin
authors.initials: M
authors.lastname: Gruebele
mesh_terms: D010582:Bacteriophage lambda / Q000378:metabolism*; D003198:Computer Simulation; D006358:Hot Temperature; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011312:Pressure; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared; D013696:Temperature; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D012097:Repressor Proteins; D014764:Viral Proteins; C076896:repressor protein C1, bacteriophage
keywords: downhill folding; fluorescence lifetime; lambda repressor; molecular dynamics simulation; thermal denaturation