Directly monitor protein rearrangement on a nanosecond-to-millisecond time-scale

Abstract

In order to directly observe the refolding kinetics from a partially misfolded state to a native state in the bottom of the protein-folding funnel, we used a "caging" strategy to trap the β-sheet structure of ubiquitin in a misfolded conformation. We used molecular dynamics simulation to generate the cage-induced, misfolded structure and compared the structure of the misfolded ubiquitin with native ubiquitin. Using laser flash irradiation, the cage can be cleaved from the misfolded structure within one nanosecond, and we monitored the refolding kinetics of ubiquitin from this misfolded state to the native state by photoacoustic calorimetry and photothermal beam deflection techniques on nanosecond to millisecond timescales. Our results showed two refolding events in this refolding process. The fast event is shorter than 20 ns and corresponds to the instant collapse of ubiquitin upon cage release initiated by laser irradiation. The slow event is ~60 μs, derived from a structural rearrangement in β-sheet refolding. The event lasts 10 times longer than the timescale of β-hairpin formation for short peptides as monitored by temperature jump, suggesting that rearrangement of a β-sheet structure from a misfolded state to its native state requires more time than ab initio folding of a β-sheet.

Figure 1

The energy landscape of protein folding and misfolding diagram. In this study, a photolabile cage, which is shown as a green ball, is used to trap a protein in a locked misfolded state and change energy landscape of folding (dashed line). After laser irradiation, the cage is photolyzed and released. The misfolded state is free to refold to the native state.

Figure 2

The schematic diagrams of two photothermal methods. (a) photoacoustic calorimetry; (b) photothermal beam deflection.

Figure 3

Structural comparison between V5C and V5C-DMC using CD, NMR and MD simulation. (a) Far-UV CD spectra of V5C and V5C-DMC. (b) Near-UV CD spectra of V5C and V5C-DMC. V5C and V5C-DMC were dissolved in DI (pH 5.7) to a final concentration of 4.76 and 6.59 μM for (a) and 40.9 and 44.2 μM for (b), respectively. (c) Hydrogen bonding networks in β-sheet of the simulated V5C-DMC structure. Red sticks: DMC. Green ribbons: β-strands. Red dotted lines: hydrogen bonds. Yellow dotted lines: hydrogen bonds lost in V5C-DMC but present in V5C. (d) Rearrangement of residues in the hydrophobic core of V5C-DMC. Green sticks: rearranged residues 43, 50 and 67 after simulation. Red sticks: DMC-conjugated Cys5 in the simulated V5C-DMC structure. Blue sticks: original positions of residues 43, 50 and 67 in V5C. If DMC was directly conjugated onto Cys5 without relaxing the structure, there is steric hindrance in residues 43, 50 and 67. The local protein surface was shown in light blue for V5C and light green for V5C-DMC. β-Strands are labeled as S1 - S5; helix 1 is labeled as H1. (e) Comparison of the 1D-NMR spectra of V5C (light blue), V5C-DMC before photolysis (green), and V5C-DMC after photolysis (red). The concentration of V5C and V5C-DMC is 175 and 20 μM, respectively. The concentration of photolytic product of V5C-DMC, i.e. refolded V5C, is 60 μM.

Figure 4

Comparison of structural stability of V5C and V5C-DMC. V5C and V5C-DMC were dissolved in DI (pH 5.7) to a final concentration of ~20 μM. (a) Wild-type ubiquitin, V5C, and V5C-DMC were digested by different concentrations of trypsin and analyzed by SDS-PAGE. (b) The thermal denaturation curves of V5C and V5C-DMC.

Figure 5

The refolding kinetic data of V5C-DMC after photolysis observed by PAC and PBD. (a) The PBD signals of reference (black), V5C-DMC (red) and V5C-DMC multiplied by 1.66 (blue). (b) The deconvolution results of PBD signals: V5C-DMC (black), the deconvoluted fast (blue) and slow (pink) events, and the fitting curve (red). (c) The PAC signals of reference (black), V5C-DMC (red), the fitting curve (blue). The corresponding residuals of fitting are shown below the plots. (d) The plots are of E _hv*ϕ versus C _p*ρ/β for the PAC signal (■), the fast event of PBD signal (V5C-DMC-fast, ●), and the slow event of PBD signal (V5C-DMC-slow, ▲).

Figure 6

Refolding reaction coordinate of the partially misfolded V5C-DMC after photolysis.