Conformation of Membrane Proteins: Bacteriorhodopsin

One of the main goals of this research project was accomplished. A water soluble derivative of bacteriorhodopsin BR was synthesized. An activated species of methoxypolyethylene glycol MeOPEG was synthesized, yielding 2-0- methoxypolyethylene glycol-N-hydroxy succinimyl carbonate. MeO-PEG-SC. MeOPEG- SC was coupled with the purple membrane PM of Halobacterium hilobium to yield MeO-PEG-PM. This product was centrifuged and purified by washing with H20 76 conversion, dissolved in buffer with 5 sodium dodecylsulphate SDS, 1 1 and PAGE Electrophoresis performed. The separated MEO-PEG-BR band was recovered from the gel by electroelution, and the solution lyophilized. This material was dissolved in H20 and the SDS removed by passing the solution through an Extra- gel column Pierce and eluted with P04 buffer pH 7.0. The final solution was centrifuged at 200,000 g, yielding a clear water-soluble solution of MeO-PEGBR. The product could be reconstituted into miscells which were capable of proton pumping. The MeO-PEG-BR reconstituted into vesicles had physical-chemical properties identical with the original PM. The circular dichroism spectra, ultraviolet and visible spectrum, and fluorescence spectrum were identical to that of the PM. Proteins, Membranes, Conformation, Bacteriorhodopsin.