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.

Keywords: downhill folding; fluorescence lifetime; lambda repressor; molecular dynamics simulation; thermal denaturation.

Fig. 1.

Pressure denaturation of λ*YA probed by fluorescence spectroscopy of a 200-μM sample (A–C) and fluorescence lifetime analysis of a 300-μM sample (D). (A) Fluorescence spectra of λ*YA in guanidine (pH 7) at 100-bar intervals from 1 to 2,500 bar (rainbow gradient). The basis spectrum of the folded state is shown in black, and that of the fully unfolded state is shown in purple (SI Appendix). a.u., arbitrary units. (B) Fluorescence peak shift (centroid) as a function of pressure. λ*YA in 2.4 M GuHCl shows a much larger 0.0032-nm/bar shift than λ*YA in buffer (native state model) or NATA (denatured state model). (C) Fraction folded was calculated by fitting the spectra in A to a linear combination of the folded and unfolded basis spectrum (two-state model; SI Appendix); at 1,200 bar (initial condition for P-jumps), ∼40% of the protein is unfolded. The crystallographic structure of λ*YA obtained from the PDB (ID code 3KZ3) is shown. (D) Scaled fluorescence lifetime change relative to NATA (1 at 1 bar, 0 at 1,200 bar). NATA and λ*YA in 0 M GuHCl lifetimes decrease linearly with pressure, whereas λ*YA in 2.4 M GuHCl shows the onset of pressure denaturation (χ for proteins was shifted up by +3 because NATA has a much longer lifetime; SI Appendix, Fig. S4).

Fig. 2.

P-jump instrument. The sample is pipetted into a dimple in a sapphire cube. The dimple is covered with mylar-coated aluminum foil and pressurized by pumping ethanol into a pressure fitting. A current burst into a copper electrode bursts the upper steel membrane and releases the pressure. Sample fluorescence is excited by a 280-nm pulsed laser every 12.5 ns and is collimated by a UV light guide onto a photomultiplier. The digitized raw data consist of a train of fluorescence decays, whose lifetime and intensity monitor the refolding of the sample after the sudden P-drop at t = 0.

Fig. 3.

P-jumps (300-μM sample) and T-jumps (200-μM sample) of λ*YA and NATA, probed by tryptophan fluorescence decays. Tryptophan lifetime change was normalized for NATA so that χ = 0 corresponds to the decay lifetime before the jump (1,200 bar) and χ(t) = 1 corresponds to the decay lifetime 5 ms after the jump (1 bar). The rest of the jumps were analyzed using the lifetime decays from the P-jump of NATA for direct comparison. Solid black curves are the double-exponential fits of the data with relaxation times τ_f = 3.8 ± 0.4 μs and τ_s = 1.4 ± 0.2 ms for the P-jump and τ_f = 63 ± 2 μs and τ_s = 2.17 ± 0.02 ms for the T-jump.

Fig. 4.

Equilibrium denaturation of λ*YA by pressure and temperature, probed by IR spectroscopy (1.7-mM sample). (A) IR absorbance spectra of λ*YA in the amide I′ region measured at 295 K. Triangles indicate 1 bar, and circles indicate 13.9 kbar. These spectra were used as basis functions for the analysis of the entire pressure denaturation curve (Methods and SI Appendix). The IR absorbance spectrum of λ*YA in the amide I′ region measured at 1 bar and 368 K is shown as a gray dashed line. (B) Denaturation of λ*YA as a function of pressure [χ(P) = 1 means the 1-bar basis function contributes 100% of the signal, χ(P) = 0 means the 13.9-kbar basis function contributes all the signal]. A thermodynamic two-state fit of the data is shown as a solid black curve, and the error bars are the residuals (SI Appendix, Fig. S5). The midpoint of pressure denaturation, P_m, is equal to 6.0 ± 0.2 kbar in the absence of denaturant.

Fig. 5.

CHARMM27 simulation of λ*YA during P-drop. (Upper) Structures from the two trajectories. The high-pressure simulations start with 1 μs at 325 K and 5 kbar (blue zone), followed by a 0.15-μs P-drop to 1 bar (white zone). (Lower) Refolding (8.85 μs) was simulated at 1 bar and 325 K. Central carbon atom root mean square displacement values were calculated relative to the crystal structure (PDB ID code 3KZ3) (13). The fraction of residues in α (gray) and β (red) conformations is shown. R_gyr is the unsolvated radius of gyration. The native mean values (green solid lines, except red for β-fraction) are from a 150-ns equilibrium simulation of the native structure at T = 325 K and P = 1 bar.

Fig. 6.

Residue-specific α-helical propensity of the simulations in Fig. 5 (black, first simulation; green, second simulation). The helical percentage was defined as the time percentage each residue spent in α-helical conformation during the last 8 μs of refolding simulation. The secondary structure of the crystal structure is shown as a color-coded background, and the sequence at the top, together with the red arrows, highlights turn/coil residues with >75% helix content in both simulations.