Transient and linear dichroism studies on bacteriorhodopsin: determination of the orientation of the 568 nm all-trans retinal chromophore

The orientation of the 568 nm transition dipole moment of the retinal chromophore of bacteriorhodopsin has been determined in purple membranes from Halobacterium halobium and in reconstituted vesicles. The angle between the 568 nm transition dipole moment and the normal to the plane of the membrane was measured in two different ways.In the first method the angle was obtained from transient dichroism measurements on bacteriorhodopsin incorporated into large phosphatidylcholine vesicles. Following flash excitation with linearly polarized light, the anisotropy of the 568 nm ground-state depletion signal first decays but then reaches a time-independent value. This result, obtained above the lipid phase transition, is interpreted as arising from rotational motion of bacteriorhodopsin which is confined to an axis normal to the plane of the membrane. It is shown that the relative amplitude of the time-independent component depends on the orientation of the 568 nm transition dipole moment. From the data an angle of 78 ° ± 3 ° is determined.In the second method the linear dichroism was measured as a function of the angle of tilt between the oriented purple membranes and the direction of the light beam. The results were corrected for the angular distribution of the membranes within the oriented samples, which was determined from the mosaic spread of the first-order lamellar neutron diffraction peak. In substantial agreement with the results of the transient dichroism method, linear dichroism measurements on oriented samples lead to an angle of 71 ° ± 4 °.No significant wavelength dependence of the dichroic ratio across the 568 nm band was observed, implying that the exciton splitting in this band must be substantially smaller than the recently suggested value of 20 nm (Ebrey et al., 1977).The orientation of the 568 nm transition dipole moment, which coincides with the direction of the all-trans polyene chain of retinal, is not only of interest in connection with models for the proton pump, but can also be used to calculate the inter-chromophore distances in the purple membrane.