TRANSIENT CHANNEL-OPENING IN BACTERIORHODOPSIN - AN EPR STUDY

Active translocation of ions across membranes requires alternating acc ess of the ion binding site inside the pump to the two membrane surfac es. Proton translocation by bacteriorhodopsin (bR), the Light-driven p roton pump in Halobacterium salinarium, involves this kind of,a change in the accessibility of the centrally located retinal Schiff base. Th is key event in bR's photocycle ensures that proton release occurs to the extracellular side and proton uptake from the cytoplasmic side. To study the role of protein conformational changes in this reprotonatio n switch, spin labels were attached to pairs of engineered cysteine re sidues in the cytoplasmic interhelical loops of bR. Light-induced chan ges in the distance between a spin label on the EF interhelical loop a nd a label on either the AB or the CD interhelical loop were observed, and the changes were monitored following photoactivation with time-re solved electron paramagnetic resonance (EPR) spectroscopy. Both distan ces increase transiently by about 5 Angstrom during the photocycle. Th is opening occurs between proton release and uptake, and may be the co nformational switch that changes the accessibility of the retinal Schi ff base to the cytoplasmic surface after proton release to the extrace llular side. (C) 1997 Academic Press Limited.