Evidence for chromophore-chromophore interactions in the purple membrane from reconstitution experiments of the chromophore-free membrane.

We recently presented evidence showing that the visible CD spectrum of the purple membrane from Halobacterium halobium consists of two contributions: a broad positive band centered at the absorption maximum due to the interaction of the chromophore with the protein to which it is bound, and an exciton coupling band due to the interaction between chromophores of adjacent bacteriohodopsin molecules in the hexagonal surface lattice (Heyn et al., 1975); This interpretation receives strong support from the present experiments in which the chromophore-free membrane is reconstituted by the addition of retinal. Since the coupling signal arises from the interaction between pairs of neighboring chromophores, its contribution to the spectrum would be expected to be very small in the initial stages of the titration experiment, but increasing quadratically with the percentage reconstitution. The broad positive band, on the other hand, is expected to increase linearly with the percentage reconstitution. On the basis of these considerations a satisfactory explanation of the CD reconstitution experiments could be given. Since it appears to be impossible to explain the titration experiments without the quadratic term, we conclude that chromophore-chromophore interactions play an important role. No significant changes in secondary structure upon reconstitution should be detected consistent with our binding model which neglects cooperativity;