Orientation of the protonated retinal Schiff base group in bacteriorhodopsin from absorption linear dichroism

Abstract

Linear dichroism experiments are performed on light-adapted bacteriorhodopsin (BR568) films containing native retinal (A1) and its 3,4-dehydroretinal (A2) analogue to measure the angle between the chromophore transition dipole moment and the membrane normal. QCFF/pi calculations show that the angle between the transition moment and the long axis of the polyene is changed by 3.4 degrees when the C3-C4 bond is unsaturated. The difference vector between the two transition moments points in the same direction as the Schiff base (N----H) bond for the all-trans BR568 chromophore. Because the plane of the chromophore is perpendicular to the membrane plane, a comparison of the transition moment orientations in the A1- and A2-pigments enables us to determine the orientation of the N----H bond with respect to the absolute chromophore (N----C5 vector) orientation. The angles of the transition moments are 70.3 degrees +/- 0.4 degrees and 67.8 degrees +/- 0.4 degrees for the A1- and A2-pigments, respectively. The fact that the change in the transition moment angle (2.5 degrees) is close to the predicted 3.4 degrees supports the idea that the chromophore plane is nearly perpendicular to the membrane plane. The decreased transition moment angle in the A2-analogue requires that the N----H bond and the N----C5 vector point toward the same membrane surface. Available results indicate that the N----C5 vector points toward the exterior in BR568. With this assignment, we conclude that the N----H bond points toward the exterior surface and its most likely counterion Asp-212. This information makes possible the construction of a computer graphics model for the active site in BR568.

Light-induced isomerization causes an increase in the chromophore tilt in the M intermediate of bacteriorhodopsin: a neutron diffraction study.

“…On the basis of the measured distance between the two projected label positions in BR568, the polyene chain is at a 240 angle to the plane ofthe membrane, assuming a straight all-trans polyene chain and taking into account that the line between the two centers of deuteration (0) is at a 150 angle to the polyene chain. This value is slightly larger than the values of =200 for the transition dipole moment (22,23). The situation is presented in Fig.…”

Replacement of leucine-93 by alanine or threonine slows down the decay of the N and O intermediates in the photocycle of bacteriorhodopsin: implications for proton uptake and 13-cis-retinal----all-trans-retinal reisomerization.

“…The lines of evidence leading to these conclusions are as follows: (i) The chromophores of the L93A and L93T mutants are blue-shifted What is the specific nature of the interaction of Leu-93 with retinal? A possible mechanism for this interaction is suggested by the combined structural evidence from recent cryoelectron microscopic studies (16), by the orientation of the ionone ring of retinal inferred from linear dichroism (26) and neutron diffraction (27) experiments, and by the RR experiments presented here. In a molecular model of bR based on the coordinates provided by R. Henderson et al (16), Leu-93 is seen to be in van der Waals contact with the methyl group on C-13 ofretinal.…”

Electron-crystallographic Refinement of the Structure of Bacteriorhodopsin

“…b Linear dichroism measurements (Lin & Mathies, 1989). The transition dipole of the long axis of retinal may not exactly coincide with the C 5 -C 15 vector, and the actual angle of the C 5 -C 15 vector may, therefore, be slightly larger than measured.…”