The chromophore retinal hinders passive proton/hydroxide ion translocation through bacteriorhodopsin.
Experiments have been performed to examine any influence of the chromophore retinal in bacteriorhodopsin (BR) on the passive proton/hydroxide ion flux through this integral membrane protein. BR was reconstituted into dimyristoylphosphatidylcholine (DMPC)-phosphatidylserine or DMPC-dimyristoylphosphatidylglycerol unilamellar vesicles with molar lipid to protein ratios ranging from 30 to 150. The entrapped fluorescence dye pyranine served as a reliable indicator of the internal proton concentration. Transmembrane pH-gradients were quickly established across the vesicular membrane and the kinetics of the induced fluorescence changes were compared for vesicles with incorporated native BR, BR bleached to the chromophore-free protein bacterioopsin, and BR regenerated from bacterioopsin with all-trans-retinal, respectively. For aggregated protein molecules, the H+/OH- diffusion across bacterioopsin was always considerably faster than that through the protein containing covalently bound retinal. The decay rate of the imposed pH-gradient was 4.4-9.1 and 2.0-5.1 times slower for native and regenerated BR, respectively, as compared to bacterioopsin. Stepwise regeneration of bacterioopsin with all-trans-retinal revealed a linear dependence of the predominant delta pH-decay time on the degree of regeneration. Essentially the same observations were made with monomeric protein molecules in vesicular lipid membranes. The results demonstrate that the chromophore retinal itself blocks the H+/OH- conducting pathway across the transmembrane protein BR or indirectly controls this path by inducing conformational changes in the protein upon binding.
Burghaus
P A
PA
Department of Physics, Freie Universität Berlin, Federal Republic of Germany.
Dencher
N A
NA
D001436:Bacteriorhodopsins / Q000378:metabolism*; D004134:Dimyristoylphosphatidylcholine; D006217:Halobacterium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006878:Hydroxides / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D010718:Phosphatidylserines; D011522:Protons; D012172:Retinaldehyde / Q000494:pharmacology*; D012176:Retinoids / Q000494:pharmacology*
D006878:Hydroxides; D008081:Liposomes; D046911:Macromolecular Substances; D010718:Phosphatidylserines; D011522:Protons; D012176:Retinoids; D001436:Bacteriorhodopsins; C031356:hydroxide ion; D012172:Retinaldehyde; D004134:Dimyristoylphosphatidylcholine