VECTORIALLY ORIENTED FIXATION OF MEMBRANE-EMBEDDED BACTERIORHODOPSIN ONTO AN INERT BASE

This paper describes a novel surface processing technique aimed at the chemical fixation of proteins on substrate surfaces. The essential fe ature of this newly developed technique is a combination of chemical a nd enzymatic processes. A simple technique using chemical modification and selective enzymatic digestion for immobilizing biomembrane-embedd ed proteins on inorganic solid bases, while strictly regulating their vectorial orientation, was developed. These processes are applicable t o a wide range of membrane-embedded and individual proteins, because t hey exploit the most fundamental principle of proteins, that any prote in has at least one N-terminus and one C-terminus. After thorough prot ection of the carboxyl and amino groups on the molecular surface of ba cteriorhodopsin (bR) embedded in purple membrane (PM), in which the bR molecules are uniformly oriented, the derivatized bR was subjected to successive enzymatic digestions to regenerate the unique N-terminal a mino group on the molecular surface. The derivatized bR was anchored o n an inorganic base by the regenerated amino groups and formed an orie nted layered structure. This was proved by analyzing the distance from the base to the gold clusters marking the enzymatically exposed C-ter mini, which was 4.4 nm, as measured by the fluorescent X-ray interfere nce pattern. This thickness coincides well with that of native PM with embedded bR (4 mn). From the viewpoint of its simplicity, this immobi lization process might have an advantage over the former multistep pro cesses for surface functionalization and those for the regulation of t he molecular orientation of proteins.