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

We determined the change in orientation of the electronic transition dipole moment with respect to the membrane normal in the K, L, and M intermediate s of bacteriorhodopsin using transient linear dichroism. Purple membranes w ere oriented in a 14 T magnetic field and immobilized in a gel. The oriente d purple membranes were excited isotropically and the transient absorbance changes were detected with the sample between two parallel polarizers. The absorbance changes were measured as a function of wavelength, time, and ang le between the orientation axis and polarizer direction. In this way, the t ransient changes in isotropic absorbance, linear dichroism, and linear bire fringence were determined with high accuracy. The Kramers-Kronig transform of the transient linear dichroism was in excellent agreement with the trans ient linear birefringence and this served as a useful control on the reliab ility of the linear dichroism data. We developed a novel formalism to extra ct the anisotropies, spectra and time courses of the photocycle intermediat es in a model-independent way from the combined analysis of the transient a bsorbance and linear dichroism data. Whereas an analysis based on transient absorbance data alone is underdetermined, we show that in combination with transient linear dichroism data a unique solution may be obtained for the early intermediates K, L, and M. The analysis makes use of the constraints that (1) the sum of the populations of the K, L, and M intermediates is con stant in time and (2) the absorption for the M intermediate vanishes for la mbda greater than or equal to 520 nm. For wild-type bR at pH 7 (10 degrees C) we obtained in this way the following wavelength-independent anisotropie s for the main absorption band: r(bR) = -0.145, r(K) = -0.140, r(L) = -0.13 2, and r(M) = -0.139. Similar experiments were carried out for the mutant D 96A which allows more accurate experiments for the L and M intermediates un der various conditions of temperature and pH (pH 7, 20 degrees C; pH 4.7, 2 0 degrees C; pH 4.7, 10 degrees C). In all cases there are very clear diffe rences in the anisotropies and the sequence is always r(bR) < r(K) < r(M) < r(L). The data analysis is validated by the fact that the spectra and time courses of the intermediates are in excellent agreement with previous work . Making the reasonable assumption that the order parameter characterizing the orientational distribution is the same for each intermediate, the aniso tropy changes translate into small orientational changes for the transition dipole moment: Delta theta(K) = -0.8 + 0.2 degrees, Delta theta(L) = -1.7 +/- 0.2 degrees, Delta theta(M) = -1.1 +/- 0.3 degrees. The largest change occurs in the L intermediate. The angle with respect to the membrane normal is smaller in every intermediate than in the ground state. The simplest in terpretation of the results is that after the isomerization of the C-13-C-1 4 double bond the C-5-N direction remains approximately the same with the C -5-C-13 part of the polyene chain tilting out of the plane of the membrane.