BINDING OF ALL-TRANS-RETINAL TO THE PURPLE MEMBRANE - EVIDENCE FOR COOPERATIVITY AND DETERMINATION OF THE EXTINCTION COEFFICIENT

At low bacteriorhodopsin concentration the binding of all-trans-trans to the apomembrane of Halobacterium halobium, as monitoried by the absorbance change at 568 nm, occurs in a cooperative manner. The simplest way of analyzing the binding data is based on an all-or-none model and results in a Hill coefficient of 3.0 ± 0.2 and an apparent association constant of 2.8 X 106 M-1. The same sigmoidal binding curve was obtained by using the change in circular dichroism at 365 nm (displacement of retinal oxime) or at 263 nm (retinal-induced change in bacterioopsin). Moreover, the trivial explanation of our results, namely, that the sigmoidal shape is caused by a suitably varying extinction coefficient, could be excluded, since the extinction coefficient was shown to be independent of the degree of binding. The Hill coefficient of close to 3 suggests that protein-protein interactions within bacteriorhodopsin trimers are responsible for the observed cooperative effect. Such an interpretation is consistent with the structure of the reconstituted apomembrane which consists of a hexagonal lattice of bacteriorhodopsin in which the bacteriorhodopsin molecules are arranged in clusters of three. The surprisingly small value of the association constant shows that retinal binding to the apomembrane is not irreversible. This was confirmed by exchange experiments between retinal 1 and retinal2 which show that bound retinal2 can be displaced by retinal! and vice versa. At bacteriorhodopsin concentrations much higher than the reciprocal of the association constant, all the retinal added is bound until all the binding sites are occupied. It is therefore possible to determine the extinction coefficient of the chromophore from the slope of the binding curve. The extinction coefficient obtained in this way is based on a knowledge of the retinal concentration and does not depend on a determination of the protein concentration. The resulting value of 62 700 ± 700 M-1 cm-1, at 568 nm, refers to the light-adapted state of the purple membrane at 25 °C in 0.02 M phosphate buffer, pH 6.9, and is corrected for light scattering. © 1979, American Chemical Society. All rights reserved.