TIME-RESOLVED POLARIZED ABSORPTION-SPECTROSCOPY WITH ISOTROPICALLY EXCITED ORIENTED PURPLE MEMBRANES - THE ORIENTATION OF THE ELECTRONIC-TRANSITION DIPOLE-MOMENT OF THE CHROMOPHORE IN THE O-INTERMEDIATE OF BACTERIORHODOPSIN

The chromophore of the retinal-protein bacteriorhodopsin undergoes Lig ht-induced isomerization around the 13-14 double bond and senses prote in conformational changes. These events are accompanied by changes in the orientation of the electronic transition dipole moment and may be conveniently monitored by time-resolved polarized absorption spectrosc opy. We used samples of oriented immobilized purple membranes and exci ted them isotropically. This has a number of advantages over photosele ction with suspensions of membranes: (1) a high signal to noise ratio, (2) absence of depolarization due to rotational diffusion, and (3) pr ecise determination of the angle of the transition dipole moment and t he membrane normal. We show that with isotropic excitation the anisotr opy of a transient intermediate i is given by r(i) = S2P2(cos theta(i) ), in which S-2 is the orientational order parameter of the membranes and theta(i) the angle of the transition dipole moment with respect to the membrane normal. S-2 may be obtained from the steady-state anisot ropy of the initial state. This approach was applied to determine the transition dipole orientation in the O-intermediate. Purple membranes were oriented in a 14-T magnetic field and immobilized in a polyacryla mide gel. S-2 was 0.74. The polarized absorbance changes were measured on a quasi-logarithmic time scale from 100 ns to 10 s at 16 wavelengt hs after isotropic excitation with a 20-ns flash. The anisotropy of th e depletion signal of isotropic purple membrane gels was measured as a control to check if the excitation was isotropic and was found to be zero over more than four decades of time. With oriented gels the aniso tropy data were analyzed for the O-intermediate both from the time tra ces and from the amplitude spectra associated with the rise and decay in a global exponential fit. Both methods lead to an anisotropy for O of about -0.23, which does not depend on a model for the photocycle ki netics. We conclude that the angle between the transition dipole momen t and the membrane normal in O (all-trans, 640 nm) is about 2 degrees larger than in the initial state (all-trans, 568 nm).