The purple membrane (PM) from H. Salinarium contains a single, well defined, 26000 D protein (bacteriorhodopsin (bR)) in intimate association with 10 lipid molecules. The monomer is part of a trimer arranged in a symmetric, hexagonal, two dimensional crystal. PM performs the same function as a respiratory chain in its ability to transduce an input energy into a electrochemical proton gradient used to form ATP. Our laboratory has shown that specific lipids of the membrane control the conformational flexibility of bR alpha-helices and the photocycle itself. Specifically, squalene appears to mitigate a repulsive interaction of polar lipids with aspartates in the cytoplasmic loop region to lesson a contortional strain which decreases the kinetics of the photocycle. Our laboratory has challenged the prevailing view that the photocycle is an homogeneous series of reversible linear conversions of intermediates (RHM) represented as BR <-> K <-> L <-> M1 <-> M2 <-> N <-> O <-> BR. In this view, the M1 -> M2 conversion represents an essential gate for proton transport where the retinal in a Schiff base with lys-216 shifts its orientation from a cytoplasmic channel to an outside channel. Extensive X-ray diffraction evidence has been produced to support this view. However, our laboratory has shown that the bR photocycle, at pH 7 and 20 degrees C, is better represented by separate unidirectional parallel cycles (UPM) , specifically, BR -> K -> L -> M1 -> N -> O -> BR, and BR ->K -> L -> M2 -> BR. If the M1 and M2 are in separate cycles, the proton gate model can not be true. This past year, we have shown that the UPM fits experimental data both from our laboratory and others better than does the RHM. We also have established the universality of the UPM in its ability to deconvolute raw data collected under nine conditions of varied temperature and pH. In this survey, we have found two additional photocycles that become prominent at different pH?s and T' s: BR -> K -> L -> M -> O -> BR, and BR -> K -> L -> M -> N -> BR. If one plots the the locations of cycles on a grid of pH in one dimension and T in the other, a phase-like diagram is seen in which different combinations of the four basic cycles are found in common areas of pH and T. This contradicts the current view that only a single RHM exists and that its kinetics as a function of T observe the Arrhenius equation. Because the RHM and UPM are entirely distinct, the fitting of UPM to all of these different data sets strongly supports the validity of UPM unless, the UPM can unexplainably also fit data generated by a RHM. To test this possibility we synthesized data based on RHM and demonstrated that they could not be fit by a UPM. Based on our ability to extract the absolute spectrum for each intermediate from raw kinetic data, we have started a collaboration with Aleksandr Smirnov to try to obtain structures for each intermediate in the photocycle using time-resolved X-Ray diffraction data. With a simultaneous kinetic study of the magnitudes of proton current and voltage formation during each step of the photocycle we hope to learn much more about the proton-pumping process and the ability of the membrane to control it.
