Direct observation of fast protein conformational switching

Abstract

Folded proteins can exist in multiple conformational substates. Each substate reflects a local minimum on the free-energy landscape with a distinct structure. By using ultrafast 2D-IR vibrational echo chemical-exchange spectroscopy, conformational switching between two well defined substates of a myoglobin mutant is observed on the approximately 50-ps time scale. The conformational dynamics are directly measured through the growth of cross peaks in the 2D-IR spectra of CO bound to the heme active site. The conformational switching involves motion of the distal histidine/E helix that changes the location of the imidazole side group of the histidine. The exchange between substates changes the frequency of the CO, which is detected by the time dependence of the 2D-IR vibrational echo spectrum. These results demonstrate that interconversion between protein conformational substates can occur on very fast time scales. The implications for larger structural changes that occur on much longer time scales are discussed.

Fig. 1.

Structure of CO-bound myoglobin. (A) Crystal structure of MbCO taken from the Protein Data Bank (ID code 1MWC). The distal histidine, His-64; the CO; the location of Leu-29; and the E helix are indicated. (B) FT-IR spectrum of the CO stretch of heme-ligated CO for the myoglobin mutant L29I (solid curve). The spectrum was fitted by two Gaussians (dashed curves), which represent the absorption bands of the A₁ and A₃ substates.

Fig. 2.

2D-IR spectra of CO bound to L29I. (A) 2D-IR spectra of L29I-CO at various times, T_w. Each contour corresponds to a 10% signal change. The bands in the upper half of the spectrum (positive-going) correspond to the 0–1 vibrational transition. The bands in the lower half of the spectrum (negative-going) arise from vibrational echo emission at the 1–2 transition frequency. (B) Calculated 2D-IR spectra of L29I-CO at various times, T_w, using the known input parameters and the substate switching time constant obtained from fitting the data.

Fig. 3.

Peak volume data obtained from the fitting of the 2D-IR spectra of L29I-CO for the 0–1 transition region. The diagonal A₁ and A₃ bands as well as the off-diagonal peaks that grow in because of substate switching are fit with a single adjustable, the substate switching time, τ₁₃ = 1/k₁₃. The solid curves are the results of the fits, which yield τ₁₃ = 47 ps.

Fig. 4.

Schematic illustration of a portion of the energy landscape of a protein, showing two substates and the finer energy landscape that exist in each of the minima. Transitions among the minima in one substate are responsible for structural fluctuations about the substate minimum. Transitions from one substate to another represent distinct structural configurational changes of the protein.