Recent findings in the structure—functional characteristics of bacteriorhodopsin

Cooperativity in the photocycle of purple membrane of Halobacterium halobium with a mechanism of free energy transduction

“…Furthermore, the chemical sequence [9] suggests additional interactions through terminal peptide chains as well as amino acid residues. A consequence of these interactions is the complete immobilization of the protein within the purple membrane, allowing no rotational freedom to bacteriorhodopsin even in the minute time range [8].…”

The antigenic structure and topography of bacteriorhodopsin in purple membranes as determined by interaction with monoclonal antibodies

“…The interaction of mAbs with the purple membrane (PM), bacterioopsin (BO) and fragments of BR (see table 2) was tested by ELISA; ( + ) positive interaction, ( -) negative interaction mAb ID2Gi interaction with PM was completely abrogated after treatment of membranes with papain (as in [8]), resulting in cleavage of regions l-3, 66-72 and 232-248 from the protein molecule (table 4). Since, as shown above, the epitope for mAb IDzGi is located within the region l-20, this result means that at leasts one of 3 removed residues, <Glu', Ala2 or Gln3, is a part of the epitope.…”

“…After centrifugation (4 times) in HzO, the membranes were treated with papain to remove C-terminal fragment 232-248 . [8]. Peptide 156-231 was isolated by Sephadex LH-60 chromatography in 98% formic acid-absolute ethanol (3:7).…”

“…Papain cleavage of BR in PM was carried out according to [8]. To modify carboxyl groups [22], the PM (1 mg/ml) were dissolved in 5 ml of 0.2 M sodium phosphate buffer, pH 4.75, containing 2 M ethylenediamine, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added to a final concentration of 0.4 M. The pH value was carefully controlled as carbodiimide was added over a 1 h interval and the reaction allowed to proceed for a further 5 h. After the reaction was completed, the membranes were spun down in water.…”

Chemical modification of purple membranes: role of arginine and carboxylic acid residues in bacteriorhodopsin

“…In general, carbodiimide-catalyzed amide formation results in an 0-acyl isourea intermediate which in aqueous solution, either condenses with amines to yield corresponding amides, rearranges to form a more stable N-acylurea, or slowly hydrolyzes to regenerate the carboxyl group [21]. Kinetic studies on model carbodiimide-carboxyl-nucleophile systems have shown that rearrangement can be rendered slow compared to nucleophile attack if the concentration of nucleophile is sufficiently high [22].…”