Bacteriorhodopsin (bR), a light-driven proton pump, consists of a bundle of seven membrane-spanning alpha-helices connected to each other by short extramembranous loops. Previously it has been shown that bR can be reconstituted from three fragments corresponding to the first helix, the second helix, and the remaining five helices, and that this reconstituted material reforms the native structure of bR. In this study, it is shown that the native function is also recovered. Low-temperature spectroscopy was used to examine the photochemical properties of bR reconstituted from three fragments. At room temperature at pH 6, the reconstituted material shows essentially the same absorption spectrum as native bR, while upon raising the pH at room temperature or cooling the sample in glycerol, a second, blue-shifted peak appears. The pH and temperature dependence of the absorption spectrum indicates that the reconstituted bR is in an equilibrium between two pigments, which we call P560 and P480. Both pigments convert to their own K intermediates, which differ in absorption maxima, upon illumination with green light at -180 degrees C. Each K intermediate can be reverted to its initial state by light. Similarly, both pigments convert to their own M intermediates upon irradiation with yellow light at -77 degrees C. The M intermediate of both species can be reverted only to P560 by light. Both pigments are therefore photoactive. These unique photochemical properties of bR reconstituted from three fragments may be attributable to the lack of a covalent linkage in the loop connecting the A and B helices, and thus possibly to a change in the orientation of the B helix.

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Langmuir-Blodgett (LB) films of retinoic acid and LB films of retinoic acid mixed with a peptide that contains an alaninelysine-valine (AKV) amino acid sequence deposited on a hydrogenated amorphous silicon (a-Si:H) film prepared by electron cyclotron resonance (ECR) plasma sputtering were fabricated, and their light absorption spectrums were compared. A specific visible light absorption at approximately 500 nm occurred in a film that had a film thickness of more than 80 nm and a hydrogen concentration of more than 20% in the sputtering process gas. Mixing the AKV sequence peptide with retinoic acid caused a 6 nm blueshift, from 363 to 357 nm, of the absorption maximum of the composite LB film on a SiO 2 substrate. Using the same peptide, a large 30 nm blueshift, from 500 to 470 nm, was induced in the composite LB film on the a-Si:H film.

A surface structural comparison of composite film of bacteriorhodopsin (BR) and phosphatidylcoline (PC) fabricated on a layered fatty acid film on a substrate of amorphous silicon dioxide (a-SiO 2 ), crystal silicon dioxide (c-SiO 2 ), or hydrogenated amorphous silicon (a-Si:H) was conducted to investigate the effect of substrate structure on the Langmuir-Blodgett (LB) film fabrication of membrane protein by direct force microscopy (DFM) measurement. On the a-SiO 2 substrate, a hexagonal crystal structure of BR and PC with a size of 0.5 mm was observed. However, on the c-SiO 2 or a-Si:H substrate, the surface structural features of the composite LB films differed from each other on the shape of assemblies in themselves. In spite of the presence of fatty acid layers, the assemby of BR and PC depends on the structure of the bottom substrate.

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