TY - JOUR
T1 - Misplaced helix slows down ultrafast pressure-jump protein folding
AU - Prigozhin, Maxim B.
AU - Liu, Yanxin
AU - Wirth, Anna Jean
AU - Kapoor, Shobhna
AU - Winter, Roland
AU - Schulten, Klaus
AU - Gruebele, Martin
PY - 2013/5/14
Y1 - 2013/5/14
N2 - Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein YA, the Y22W/Q33Y/G46,48A mutant of 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-coilrich 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 a-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of YA contains mainly residual helix and little β-sheet; following a fast P-drop, at least some λYA 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.
AB - Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein YA, the Y22W/Q33Y/G46,48A mutant of 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-coilrich 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 a-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of YA contains mainly residual helix and little β-sheet; following a fast P-drop, at least some λYA 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.
KW - Downhill folding
KW - Fluorescence lifetime
KW - Lambda repressor
KW - Molecular dynamics simulation
KW - Thermal denaturation
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U2 - 10.1073/pnas.1219163110
DO - 10.1073/pnas.1219163110
M3 - Article
C2 - 23620522
AN - SCOPUS:84877083850
SN - 0027-8424
VL - 110
SP - 8087
EP - 8092
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 20
ER -