PHOTOSELECTION AND TRANSIENT LINEAR DICHROISM WITH ORIENTED IMMOBILIZED PURPLE MEMBRANES - EVIDENCE FOR MOTION OF THE C(20)-METHYL GROUP OF THE CHROMOPHORE TOWARDS THE CYTOPLASMIC SIDE OF THE MEMBRANE

We have determined the transition dipole moment orientation of the chromophore during the photocycle of bacteriorhodopsin by photoselection and time-resolved linear dichroism experiments with samples of oriented immobilized purple membranes. This technique offers two important advantages over experiments with isotropic aqueous suspensions: (1) the depolarization due to the rotational diffusion of the membranes is eliminated, (2) the sensitivity for detecting the orientation of the transition dipole moment of intermediates is greatly increased. The appropriate equations for the analysis of time-resolved linear dichroism experiments with samples of oriented immobilized membranes will be presented. In the transition from the ground state of bacteriorhodopsin to the M-intermediate, the transition dipole moment tilts out of the plane of the membrane by about 3-degrees. On the basis of current structural information on the plane of the chromophore and the orientation of its C(19) and C(20) methyl groups, a tilt of the transition dipole moment into the plane of the membrane would have been expected if it is assumed that the orientation of the conjugated polyene chain from C(5) to C(13) is the same in both states. The experimental result may be explained by an 11-degrees tilt of the C(5) to C(13) part of the chain out of the plane of the membrane with the C(20) methyl group moving towards the cytoplasmic side of the membrane by about 1.7 A and the cyclohexene ring staying fixed. This interpretation is supported by recent neutron diffraction experiments on the chromophore position in the M-intermediate.