Linear Dichroism Measurements on Oriented Purple Membranes between Parallel Polarizers:  Contribution of Linear Birefringence and Applications to Chromophore Isomerization

Abstract

Absorption measurements with linearly polarized light were performed with oriented purple membranes in the wavelength range from 350 to 750 nm. The membranes were oriented in a magnetic field and subsequently immobilized in a gel. Alternatively orientation was achieved by anisotropic swelling of a dehydrated gel. This new method of orientation, which is similar to the gel-squeezing technique, yields a negative second-order parameter S 2 (e.g., the membranes orient preferentially with their planes parallel to the direction of expansion). In contrast to standard linear dichroism measurements, we used a setup where the oriented sample is inbetween two parallel polarizers. The dependence of the transmitted intensity on the angle between the orientation axis and the polarizer direction shows that the phase difference due to linear birefringence contributes substantially to the signal. Experimental data of high accuracy were obtained by taking measurements at 14 angles from −20° to 110° in steps of 10°. Fitting of the polarization data at each wavelength yields the absorption spectrum, the anisotropy and the linear birefringence. Analogous measurements were carried out on chromophore-free samples to correct for light scattering and to extract the chromophore contribution to the linear birefringence. The chromophore part of the linear birefringence is in good agreement with numerical calculations from absorption and anisotropy via the Kramers−Kronig transform. The method was applied to investigate possible changes in transition dipole moment orientation associated with the isomerization of the chromophore in light−dark adaptation (all-trans to 13-cis) and the blue−pink transition (all-trans to 9-cis). Analysis of the anisotropy indicates that there is no change in the orientation of the transition dipole moment with respect to the orientation axis between the light and dark adapted states. Only a small increase (≈2.5°) of the chromophore angle occurs between the blue state and the pink state. Comparison of the measured birefringence changes and the calculations from absorption and anisotropy changes confirm the validity of the approach.

Structural Changes in Bacteriorhodopsin During Ion Transport at 2 Angstrom Resolution

“…In comparison, the movement of the p-ionone ring by 0.4 A toward the Schiff base is relatively minor. The measured angles between the conjugated rr system of the retinal and bilayer plane for the BR and M states are 20.2" and 29.6", respectively, which is in excellent agreement with the polarized a d d t s (21).…”

“…The disposition of the polyene chain and its changes in M as well as its angle to the membrane plane and its changes have been estimated from transient linear dichroism in the visible and the infked (21) during the photocycle. Earnest et al (21) reported the angles between the conjugated rr system of the retinal and the bilayer plane in the BR and M states to be 21" and 28", respectively. The positions of the ring and the 13-methyl group in the unphotolyzed state have been de- terminecl by neutron diffraction of protein with specifically deuterated retinals (22).…”

Reorientation of the Retinylidene Chromophore in the K, L, and M Intermediates of Bacteriorhodopsin from Time-Resolved Linear Dichroism: Resolving Kinetically and Spectrally Overlapping Intermediates of Chromoproteins

“…The scattering background was determined with an oriented sample of bleached (chromophore free) membranes. 17 2.3. Time-Resolved Polarized Absorption Spectroscopy.…”

“…We introduced a measuring scheme, which we already successfully applied to steady-state linear dichroism measurements. 17 In this method, the oriented sample is placed between two parallel polarizers. The time-dependent transmitted intensity is measured as a function of the polarization angle.…”

Kinetic multichannel spectroscopy of biological molecules: Decomposition of the spectral matrix

“…The resulting f ( t j ) values were then used to rescale the difference spectra to eliminate the effect of BR recovery, and a similar SM3 was performed on the new data matrix. The absorption spectrum of the initial state was kindly provided by Dr. Berthold Borucki 6. The scattering‐free absorption is essential to obtain the true absolute spectra of the intermediates from their difference spectra.…”