Time-resolved refractive index changes taking place during the photocycle of bacteriorhodopsin (BR) in purple membrane were studied by photothermal beam deflection (PBD) upon 8 ns pulse excitation. The PBD signal was monitored in the time range from several microseconds to 10 ms and separated into its various components originating from different physical effects (thermal, volume change, and absorbance-determined contributions).

Ligand transport through myoglobin (Mb) has been observed by using optically heterodyne-detected transient grating spectroscopy. Experimental implementation using diffractive optics has provided unprecedented sensitivity for the study of protein motions by enabling the passive phase locking of the four beams that constitute the experiment, and an unambiguous separation of the Real and Imaginary parts of the signal. Ligand photodissociation of carboxymyoglobin (MbCO) induces a sequence of events involving the relaxation of the protein structure to accommodate ligand escape. These motions show up in the Real part of the signal. The ligand (CO) transport process involves an initial, small amplitude, change in volume, reflecting the transit time of the ligand through the protein, followed by a significantly larger volume 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 ligand escape via a few specific cavities, and they suggest the existence of discrete escape pathways.

Volume changes associated with the primary photochemistry of bacteriorhodopsin (BR) were measured by temperature-dependent laser-induced optoacoustic spectroscopy (LIOAS). Excitation was performed with 8-ns flashes establishing a photoequilibrium between the BR and the K states (BR<-->hvK). The concentration of K at the end of the laser pulse, which is an important parameter for the calculation of the volume change per molecule from the LIOAS data, was determined by flash photolysis with optical detection under the specific conditions (concentration, photon density) of the LIOAS experiment. Temperature-dependent measurements yielded a linear dependency of the ratio of the optoacoustic signals for BR and for a calorimetric reference (CoCl2) with the cubic thermal expansion coefficient beta of water. From the slope of this linear ratio a contraction of 11 cm3/mol was determined.

No abstract