Location of the retinal of bacteriorhodopsin's M sub(412) intermediates by phase modulation of resonance energy transfer

A novel application of modulation excitation spectroscopy has been used to measure the position of the retinal chromophore in the M sub(412) intermediates of the bacteriorhodopsin photocycle. This technique uses phase modulation of fluorescence resonance energy transfer to determine the distance from a lipid fluorescent donor to the chromophore of a reaction intermediate in a membrane protein. In this study, bacteriorhodopsin was incorporated into asolectin vesicles labeled with a fluorescent lipid probe. Energy transfer was measured from the fluorescent probes on the surface of the membrane to the retinal in the M sub(412) intermediates. Concentrations of M sub(412) intermediates in these vesicles were varied by changing the frequncy of modulation of the actinic light that drives the photocycle. The results show that the chromophore in the slow-decayig M sub(412) intermediate is located farther from the vesicle exterior than the chromophore in the fast-decaying M sub(412) intermediate. Because bacteriorhodopsin reconstitutes into these vesicles in the opposite orientation as in the native membrane, this indicates that in the intact cell the retinal in the slow M sub(412) intermediate is located farther from the cytoplasmic surface than the retinal in the fast M sub(412) intermediate. Implications of these results for the proteon-pumping mechanism of bacteriorhodopsin are