Changes in retinal position during the bacteriorhodopsin photocycle: a resonance energy-transfer study
Distances from fluorescent lipid probes to the retinal chromophore of bacteriorhodopsin incorporated into asolectin vesicles have been measured with resonance energy transfer. Steady-state fluorescence is used to find the distance of closest approach from fluorescent lipid probes to ground-state retinal. Phase modulation of resonance energy transfer is used to find distances for retinal in bacteriorhodopsin's M intermediates. This latter technique uses an actinic light source to drive the
... to drive the photocycle while absorbance of a photocycle intermediate and fluorescence due to energy transfer to that intermediate are monitored with phase-sensitive detection. The photocycle intermediate concentration is varied by changing the frequency of modulation of the actinic light. Previous measurements of distances to the M intermediates using this technique [Hasselbacher, C . A., Preuss, D. K., & Dewey, T. G. (1986) Biochemistry 25, 668-6761 are extended by use of several different fluorescent probes allowing labeling of either the bilayer surfaces or the interior. This provides a more detailed mapping of retinal's position. Analysis of the phase modulation data is improved in several ways to obtain accurate distances for the M intermediates. These distances are compared to ground-state distances. Results show that retinal is buried more deeply in the protein when bacteriorhodopsin is in its ground state than in either M state. There is also a significant change in retinal location between the slow-decaying and fast-decaying M intermediates. These large changes in retinal's position during the photocycle demonstrate the flexibility of monomeric bacteriorhodopsin. Bacteriorhodopsin is a membrane-bound protein synthesized by the archaebacterium Halobacterium halobium. It actively transports protons across the bacterial membrane and maintains the proton gradient required for metabolic processes during oxygen deprivation.