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 recon stitute a bacteriorhodopsin (BR) model membrane which gives rise to a photosignal that is pH- and temperature-sensitive. We interpreted it a s being composed of two components: B1, a pH- and temperature-insensit ive component, and B2, a pH- and temperature-sensitive component. In a ddition, we used a modification of this technique to produce a multila yered BR thin film which gives rise to a photosignal that is much larg er in magnitude and much less sensitive to both pH and temperature. St ripping of layers from the multilayered film gives rise to a photosign al which is similar in magnitude, and in pH and temperature sensitivit y 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 f rom any B2 contribution. Stripping of layers from these films has no e ffect on the pH or temperature insensitivity. We report experimental o bservations which suggest why the pH- and temperature-sensitive B2 com ponent is eliminated in a multilayered BR thin film. Our results sugge st that the B1 signal originates from an intramorecular charge separat ion, the amplitude of which is additive as the number of layers increa ses; 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 hav e 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.