COMPONENT ANALYSIS OF THE FAST PHOTOELECTRIC SIGNAL FROM MODEL BACTERIORHODOPSIN MEMBRANES .1. EFFECT OF MULTILAYER STACKING AND PROLONGED DRYING

We used a technique originally developed by Trissl and Mental to reconstitute a bacteriorhodopsin (BR) model membrane which gives rise to a photosignal that is pH- and temperature-sensitive. We interpreted it as being composed of two components: B1, a pH- and temperature-insensitive component, and B2, a pH- and temperature-sensitive component. In addition, we used a modification of this technique to produce a multilayered BR thin film which gives rise to a photosignal that is much larger in magnitude and much less sensitive to both pH and temperature. Stripping of layers from the multilayered film gives rise to a photosignal which is similar in magnitude, and in pH and temperature sensitivity to that of a Trissl-Montal membrane. Drying of the multilayered film results in a photosignal that is totally insensitive to pH, and this photosignal can be regarded as a pure component, presumably BI, free from any B2 contribution. Stripping of layers from these films has no effect on the pH or temperature insensitivity. We report experimental observations which suggest why the pH- and temperature-sensitive B2 component is eliminated in a multilayered BR thin film. Our results suggest that the B1 signal originates from an intramorecular charge separation, the amplitude of which is additive as the number of layers increases; the B2 signal originates from the binding and release of aqueous protons that can be generated only from the top layer of the film, and so the amplitude of the B2 component is diminished relative to the B1 component in a multilayered thin film. In multilayered films that have been dried for a prolonged period, the amplitude of the B2 signal is reduced to an insignificant level, presumably because of denaturation of the protein.