Conformation of Membrane Proteins: Bacteriorhodopsin

摘要

Bacteriorhodopsin, from the purple membrane (PM) of Halobacterium halobium, waschemically modified with methoxypolyethylene glycol (MW = 5000) succinimidyl carbonate. The polyethylene glycolbacteriorhodopsin (m-PEG-SC-BR33) conjugate, containing one PEG chain, was water soluble. The secondary structure of the conjugate in water appeared partially denatured but was shown to contain alpha-helical segments by circular dichroism (CD) spectroscopy. The isolated bacteriorhodopsin conjugate, with added retinal, was refolded in a mixed detergent-lipid micelle and had an absorption maximum at 555 nm. The refolded conjugate was transferred into vesicles which pumped protons, upon illumination, as efficiently as did native BR. Modification of the PM with methoxypolyethylene glycol did not alter the native structure or inhibit proton pumping, and therefore it is suggested that the glycol polymer is present as a covalently linked moiety to residues unnecessary for proton pumping and proper folding. The site of attachment of mPEG was determined to be either at Lys 129 or Lys 159, with position Lys 129 the most probable site of attachment. The m-PEG-SC-BR33 could be stepwise refolded to the native conformation by the addition of trifluoroethanol to lower the dielectric constant, simulating the insertion of the BR into the phospholipid bilayer. Membrane proteins, Bacteriorhodopsin.