Changes in retinal position during the bacteriorhodopsin photocycle: a resonance energy-transfer study

C. A. Hasselbacher, T. G. Dewey
1986 Biochemistry  
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
more » ... 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.
doi:10.1021/bi00368a060 fatcat:6xhc6gxkovhxnm5ai56bd4ehgm