Diffractive optics-based heterodyne-detected four-wave mixing signals of protein motion: From "protein quakes" to ligand escape for myoglobin

Ligand transport through myoglobin (Mb) has been observed by using opticall y heterodyne-detected transient grating spectroscopy. Experimental implemen tation using diffractive optics has provided unprecedented sensitivity for the study of protein motions by enabling the passive phase locking of the f our beams that constitute the experiment, and an unambiguous separation of the Real and Imaginary parts of the signal. Ligand photodissociation of car boxymyoglobin (MbCO) induces a sequence of events involving the relaxation of the protein structure to accommodate ligand escape. These motions show u p in the Real part: of the signal. The ligand (CO) transport process involv es an initial, small amplitude, change in volume, reflecting the transit ti me of the ligand through the protein, followed by a significantly larger vo lume change with ligand escape to the surrounding water. The latter process is well described by a single exponential process of 725 +/- 15 ns at room temperature. The overall dynamics provide a distinctive signature that can be understood in the context of segmental protein fluctuations that aid li gand escape via a few specific cavities, and they suggest the existence of discrete escape pathways.