Visualizing breathing motion of internal cavities in concert with ligand migration in myoglobin

Abstract

Proteins harbor a number of cavities of relatively small volume. Although these packing defects are associated with the thermodynamic instability of the proteins, the cavities also play specific roles in controlling protein functions, e.g., ligand migration and binding. This issue has been extensively studied in a well-known protein, myoglobin (Mb). Mb reversibly binds gas ligands at the heme site buried in the protein matrix and possesses several internal cavities in which ligand molecules can reside. It is still an open question as to how a ligand finds its migration pathways between the internal cavities. Here, we report on the dynamic and sequential structural deformation of internal cavities during the ligand migration process in Mb. Our method, the continuous illumination of native carbonmonoxy Mb crystals with pulsed laser at cryogenic temperatures, has revealed that the migration of the CO molecule into each cavity induces structural changes of the amino acid residues around the cavity, which results in the expansion of the cavity with a breathing motion. The sequential motion of the ligand and the cavity suggests a self-opening mechanism of the ligand migration channel arising by induced fit, which is further supported by computational geometry analysis by the Delaunay tessellation method. This result suggests a crucial role of the breathing motion of internal cavities as a general mechanism of ligand migration in a protein matrix.

Fig. 1.

The crystal structures of MbCO before and after photodissociation of CO at 40 K are superimposed and shown in magenta and cyan, respectively. The molecular surface of MbCO and the surface of internal cavities are shown by the mesh in purple. The internal cavities (DP, Xe1, Xe2, Xe3, and Xe4) are also indicated by dotted lines. The electron densities of bound and photodissociated CO molecules in the DP are represented in magenta and cyan, respectively, by using a 2F_o − F_c map (contoured at 0.7 e/ų). The movement of CO, heme iron atom, His-64, Leu-29, and His-93 after photodissociation is shown by yellow and green arrows.

Fig. 2.

Photodissociation and migration of the CO molecule in Mb at 120 K. (A–D) Internal cavities and time-dependent evolution of the CO electron density in the Xe1 (A), Xe2 (B), Xe3 (C), and Xe4 (D) cavities at 120 K. The surfaces of the internal cavities are shown by the mesh in purple. The electron densities of the CO molecules in the cavities are presented by using the 2F_o − F_c map (contoured at 0.3 e/ų). (E) Visible absorption spectra of MbCO crystal measured by microspectrophotometry at 120 K. (Inset) The differential absorption spectra against the initial MbCO spectrum.

Fig. 3.

Temporal evolution and correlation of the occupancy of CO and the cavity volume. (A–C)The number of electrons integrated in each cavity and normalized volumes of the Xe1 (magenta), Xe2 (blue), and Xe4 (green) cavities at 100 K (A), 120 K (B), and 140 K (C) are shown. The number of electrons was integrated by using the CCP4 program suite (31). The volume of each cavity was calculated with the program CASTp (33) and normalized by the initial volume at time 0. (D–F) The correlation between the number of electrons and the volume of cavities Xe1 (D), Xe2 (E), and Xe4 (F) is shown.

Fig. 4.

Correlated breathing motion of the internal cavities in Mb. (A and B) Structure of MbCO at 140 K before laser illumination (magenta) (A) and after 750-min laser illumination (cyan) (B). The electron densities of the CO molecules in the Xe cavities are presented by using the 2F_o − F_c map (contoured at 0.3 e/ų). The surfaces of the internal cavities are shown by the mesh. The cavities are also outlined by dotted lines. (C) Amino acid residues lining the DP, Xe4, Xe2, Xe1, and Xe3 cavities. The color scheme is the same as that in A and B. The outlines of the cavities are also superimposed. The movements of amino acid residues between the cavities are shown by yellow arrows, and those between the Xe3 cavity and solvent area are shown by red arrows. The white arrows represent the ligand migration pathway between the cavities. (D) Strain tensors calculated by using 2 coordinates without laser illumination and after 750-min laser illumination. The strain tensors are shown with the maximum absolute eigenvalue, and the color of the segment shows the magnitude of the eigenvalue (blue, −0.20; green, 0, red, +0.20). The blue segments represent contraction, and the red segments show expansion. (E) Schematic drawing of the correlated ligand migration in a protein.