The energy landscape of myoglobin:An optical study

In this paper we demonstrate how the potential energy surface of a protein, which determines its conformational degrees of freedom, can be constructed from a series of advanced nonlinear optical experiments. The energy landscape of myoglobin was probed by studying its low-temperature structural dynamics, using several spectral hale burning and photon echo techniques. The spectral diffusion of the heme group of the protein was studied on a time scale ranging from nanoseconds to several days while covering a temperature range from 100 mK to 23 K. The spectral line broadening, as measured in three-pulse stimulated photon echo experiments, occurs in a stepwise fashion, while the exact time dependence of the line width is critically dependent on temperature. From these results we obtained the energy barriers between the conformational states of the protein. Aging time dependent hole-burning experiments show that, at 100 mK, it takes several days for the protein to reach thermal equilibrium. When, after this period a spectral hole is burned, the line broadening induced by well-defined temperature cycles is partly reversed over a period of several hours. From this we conclude that a rough structure is superimposed on the overall shape of the potential energy surface of the protein. By combining the hole burning and photon echo results, we construct a detailed image of this energy landscape, supporting the general concept of a structural hierarchy. More specifically, we show that the number of conformational substates in the lower hierarchical tiers is much lower than was previously anticipated and, in fact, is comparable to the number of taxonomic substates.