Fluorescent labeling of bacteriorhodopsin: implications for helix connections

Purple membrane from Halobacterium halobium was reacted with dansyl (5-dimethylamino-1-naphthalenyl fluorescent labels that have specificity for different protein side chains of bacteriorhodopsin. Dansyl chloride was found to react primarily with Lys-41. Dansyl hydrazine was coupled, with water-soluble carbodiimide, to Glu-74 and/or Asp-85, which was the major modified site after papain-cleavage of the carboxyl-terminal 17 amino acids. Fluorescence energy transfer was used to probe the proximity of the modified sites to the retinal chromophore of bacteriorhodopsin. The dansyl group on Lys-41 was greater than 2.99 nm from retinal, while the dansyl group on Glu-74/Asp-85 was greater than 2.10 nm from retinal. Information available on the location of retinal in the transmembrane profile and probable surface locations of the fluorescent labels was combined with the energy transfer results to calculate distances projected in the plane of the membrane. The projected distances to retinal were 1.64 nm (Lys-41) and 1.65 nm (Gly-74). These measurements, combined with many other labeling experiments that have been reported, restrict the number of likely helix-connection models to only three: EDCABGF, FEDCBAG and FGEABDC (in the nomenclature of Engelman et al. (1980) Proc. Natl. Acad. Sci. USA 77, 2023-2027).