TIME-RESOLVED VOLUME CHANGES DURING THE BACTERIORHODOPSIN PHOTOCYCLE - A PHOTOTHERMAL BEAM DEFLECTION STUDY

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-determine contributions). Separation of the three contributions was achieved by temperature-dependent PBD measurements and PBD experiments with polarized excitation and detection. This first application of the PBD method to a biological photoreceptor afforded the time-resolved volume changes taking place during the complete BR photocycle. Subsequent to the contraction of 11 cm(3)/mol for the first 200 ns (BR --> K) determined by laser-induced optoacoustics (Schulenberg, P. J.; Rohr, M.; Gartner, W.;Braslavsky, S. E. Biophys. J. 1994, 66, 838-843), two expansions of 60 (for the K --> L process) and 145 cm(3)/mol (for the L --> M process) were observed. A contraction of 185 cm(3)/mol resulted for the process M -->--> BR. Possible interpretations are given for these volume changes. The energy differences between the various transients after K resulted in negligible contributions of the thermal deactivation to the total PBD signal in every time range. This impaired the precise determination of the relative energy levels of the various intermediates.