Transient 2D IR spectroscopy of ubiquitin unfolding dynamics

Abstract

Transient two-dimensional infrared (2D IR) spectroscopy is used as a probe of protein unfolding dynamics in a direct comparison of fast unfolding experiments with molecular dynamics simulations. In the experiments, the unfolding of ubiquitin is initiated by a laser temperature jump, and protein structural evolution from nanoseconds to milliseconds is probed using amide I 2D IR spectroscopy. The temperature jump prepares a subensemble near the unfolding transition state, leading to quasi-barrierless unfolding (the "burst phase") before the millisecond activated unfolding kinetics. The burst phase unfolding of ubiquitin is characterized by a loss of the coupling between vibrations of the beta-sheet, a process that manifests itself in the 2D IR spectrum as a frequency blue-shift and intensity decrease of the diagonal and cross-peaks of the sheet's two IR active modes. As the sheet unfolds, increased fluctuations and solvent exposure of the beta-sheet amide groups are also characterized by increases in homogeneous linewidth. Experimental spectra are compared with 2D IR spectra calculated from the time-evolving structures in a molecular dynamics simulation of ubiquitin unfolding. Unfolding is described as a sequential unfolding of strands in ubiquitin's beta-sheet, using two collective coordinates of the sheet: (i) the native interstrand contacts between adjacent beta-strands I and II and (ii) the remaining beta-strand contacts within the sheet. The methods used illustrate the general principles by which 2D IR spectroscopy can be used for detailed dynamical comparisons of experiment and simulation.

Fig. 1.

Downhill unfolding dynamics. A T-jump induces a barrier shift toward the folded state. A subensemble is trapped at the shifted transition state and unfolds in a downhill manner on the nanosecond–microsecond time scale (A). The downhill unfolding appears as a burst phase in experiments with millisecond time resolution. Subsequently, the excess population in the folded well unfolds across the barrier, which results in a millisecond exponential relaxation (B). The dotted vertical lines indicate barrier positions before and after the T-jump.

Fig. 2.

Structure of ubiquitin. (a) Crystal structure of ubiquitin (30) rendered with MOLMOL (31). (b) Projection of the β-sheet of ubiquitin. A square box with a digit n represents a peptide group formed by residues n and n + 1. Red and purple lines indicate native contacts used in calculating the interstrand distance coordinates R₁ and R₂, respectively, in Fig. 6.

Fig. 3.

Equilibrium thermal unfolding of ubiquitin monitored by 2D IR spectroscopy. Parallel (ZZZZ) (a) and perpendicular (ZZYY) (b) polarization geometries. Spectra are normalized to the maximum of the 63°C spectrum. Twenty-one contours are plotted for ±60% of the spectra at 63 and 72°C and for ±15% of the difference spectra (Right). Positive and negative peaks are indicated by red and blue. Green and purple arrows represent cross-peaks. In the difference spectra, red and blue arrows indicate the diagonal peaks on the red and blue sides of the ω₃ axis, respectively. ν_⊥ and ν_‖ transitions are marked with lines.

Fig. 4.

Transient 2D IR difference spectra (ZZZZ) after a T-jump from 63 to 72°C. Transient difference spectra are plotted as a function of delay τ. Twenty-one contours are plotted at ±1.5% of the maximum of the reference spectrum at T_i = 63°C. The spectra from 1 to 7 ms are obtained from the same data set as the 100-ns spectrum. The red ellipse in Upper Left indicates depletion on the red side of the diagonal region.

Fig. 5.

Semilog plot of transient changes in 2D IR spectra. (a) Temporal profile of unfolding and refolding of ubiquitin constructed from the first SVD component of the transient difference spectra shown in Fig. 4. (b) Transient changes of slices at ω₁ = 1,642 cm⁻¹ for representative delays between τ = 100 ns (blue solid line) and 7 ms (red dashed line). (c) Relaxation profiles from ω₃ slices in b at ω₃ = 1,639 cm⁻¹ (light blue) and 1,663 cm⁻¹ (magenta). (d and e) Transient slices at ω₁ = 1,620 cm⁻¹ and the corresponding frequency shift of the positive peak ω₃*. (f) Relative changes in the antidiagonal width of the ν_⊥ (light blue) and random coil (magenta) components marked with arrows in the Inset. The normalized transient temperature relaxation profile (black dashed line) is also shown.

Fig. 6.

Thermal unfolding simulation and calculation of 2D IR spectra. (Upper Left) The unfolding simulation (46) is plotted along the coordinates R₁ and R₂ defined in the text. Protein snapshots correspond to five persistent structural regions (shown in green, red, blue, magenta, and cyan, respectively). The trajectory after a 17-ps window average is plotted in black. (Lower) Ubiquitin 2D IR spectra calculated at equilibrium (25°C) (adapted from ref. 21) and for regions D and E. The D and E difference spectra, relative to that for region A, are also shown. (Upper Right) Trends in ν_⊥ frequency and intensity and in ν_R intensity obtained from ω₁ = 1,640 cm⁻¹ and ω₁ = 1,662 cm⁻¹ slices, respectively.