Neuroglobin dynamics observed with ultrafast 2D-IR vibrational echo spectroscopy

Abstract

Neuroglobin (Ngb), a protein in the globin family, is found in vertebrate brains. It binds oxygen reversibly. Compared with myoglobin (Mb), the amino acid sequence has limited similarity, but key residues around the heme and the classical globin fold are conserved in Ngb. The CO adduct of Ngb displays two CO absorption bands in the IR spectrum, referred to as N(3) (distal histidine in the pocket) and N(0) (distal histidine swung out of the pocket), which have absorption spectra that are almost identical with the Mb mutants L29F and H64V, respectively. The Mb mutants mimic the heme pocket structures of the corresponding Ngb conformers. The equilibrium protein dynamics for the CO adduct of Ngb are investigated by using ultrafast 2D-IR vibrational echo spectroscopy by observing the CO vibration's spectral diffusion (2D-IR spectra time dependence) and comparing the results with those for the Mb mutants. Although the heme pocket structure and the CO FTIR peak positions of Ngb are similar to those of the mutant Mb proteins, the 2D-IR results demonstrate that the fast structural fluctuations of Ngb are significantly slower than those of the mutant Mbs. The results may also provide some insights into the nature of the energy landscape in the vicinity of the folded protein free energy minimum.

Fig. 1.

Crystal structure of the active site of CO-bound ferrous mouse Ngb (gray) and L29F Mb (blue) mutant protein taken from the Protein Data Bank. The heme and some selected amino acid residues are shown.

Fig. 2.

Normalized FTIR spectra of the CO stretching mode bound to Ngb (A) and Mb mutant proteins L29F (solid curve) and H64V (dashed curve) (B).

Fig. 3.

2D-IR vibrational echo spectra of NgbCO. (A) 2D-IR spectra at various times, T_w. Each contour corresponds to a 10% signal change. The red bands (positive going) correspond to the 0–1 vibrational transition. The blue bands (negative going) arise from vibrational echo emission at the 1–2 transition frequency. The dashed line in Upper Left is the diagonal. (B) Diagonal slice spectra of CO-bound Ngb at T_w = 0.25 ps (dashed curve) and 50 ps (solid curve).

Fig. 4.

2D-IR spectra of CO bound to L29F Mb mutant protein. Each contour corresponds to a 10% signal change. The dashed lines are the center lines.

Fig. 5.

T_w-dependent CLS for N₀ conformer of Ngb (A, filled triangles) and N₃ conformer of Ngb (A, filled circles) and for Mb mutant proteins H64V (B, open triangles) and L29F (B, open circles).