Conformational Substates of Myoglobin Intermediate Resolved by Picosecond X-ray Solution Scattering

Abstract

Conformational substates of proteins are generally considered to play important roles in regulating protein functions, but an understanding of how they influence the structural dynamics and functions of the proteins has been elusive. Here, we investigate the structural dynamics of sperm whale myoglobin associated with the conformational substates using picosecond X-ray solution scattering. By applying kinetic analysis considering all of the plausible candidate models, we establish a kinetic model for the entire cycle of the protein transition in a wide time range from 100 ps to 10 ms. Four structurally distinct intermediates are formed during the cycle, and most importantly, the transition from the first intermediate to the second one (B → C) occurs biphasically. We attribute the biphasic kinetics to the involvement of two conformational substates of the first intermediate, which are generated by the interplay between the distal histidine and the photodissociated CO.

Figure 1

Time-resolved difference X-ray solution scattering curves, ΔS(q,t), measured for a solution sample of wild-type sperm whale MbCO. The time delay after photoexcitation is indicated above each curve. Experimental curves (black) are compared with theoretical curves (red) that were generated from linear combinations of four time-independent species-associated difference scattering curves extracted from the kinetic analysis using the model shown in Figure 3a.

Figure 2

The result of SVD analysis in two different time ranges. (a) The first four lSVs in the entire time range (100 ps–10 ms) are shown. Four singular components of significant amplitudes were identified in the entire time range. The third and the fourth lSVs are rather noisy, but they clearly have distinct oscillatory features, as can be seen in the comparison with the first lSV (red dotted lines) superimposed on them. (b) The first four lSVs in the reduced time range (100 ps–3.16 ns) are shown. Two singular components of significant amplitudes were identified in this reduced time range.

Figure 3

The result of kinetic analysis using an optimum kinetic model. (a) The optimum kinetic model that best describes the structural dynamics of wild-type sperm whale MbCO induced by CO photolysis. (b) Population changes of the four intermediates with respect to the pump–probe time delay. The lines correspond to the populations obtained from the kinetic analysis of the experimental data, and the symbols correspond to the optimized populations at the time delay points where experimental data were measured.

Figure 4

Detailed scheme for the photoreaction of wild-type sperm whale MbCO. In particular, the movements of the CO molecule are highlighted. Yellow spheres indicate the locations of the CO for A, B, C, D, and S states. The CO ligated to the heme (A) is dissociated by the laser pulse centered at 532 nm. The photodissociated CO moves from the primary docking site (B) via the Xe4 site (C) to the Xe1 site (D) and finally to the solvent environment (S). The interplay between the distal histidine and the CO generates the conformational substates of A and B. In particular, due to the existence of the two conformational substates of B, the transition from B to C exhibits biphasic kinetics.