Site-directed fluorogenic modification of bacteriorhodopsin by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole.
Site-directed covalent modification of bacteriorhodopsin is achieved by reacting the hydrophobic probe 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) at neutral pH with purple membranes. The bacteriorhodopsin fluorescence thus produced is specific for a nucleophilic group. The spectral properties of NBD-modified bacteriorhodopsin indicate covalent interaction of the probe with the nucleophilic epsilon-amino group of a lysine residue. Modification of tyrosine can be excluded. As demonstrated by polypeptide fragmentation and subsequent sequence analysis, NBD binding is confined to lysine 41 within the primary structure of bacteriorhodopsin. Collisional fluorescence quenching with iodide demonstrates that, in NBD-treated purple membranes, the covalently bound label is not accessible in the aqueous phase. A hydrophobic location for the introduced fluorophor is thereby implied.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology*; D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D002338:Carotenoids / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D008565:Membrane Proteins / Q000302:isolation & purification; D010069:Oxadiazoles / Q000494:pharmacology*; D010446:Peptide Fragments / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D008565:Membrane Proteins; D010069:Oxadiazoles; D010446:Peptide Fragments; D002338:Carotenoids; D001436:Bacteriorhodopsins; D009327:4-Chloro-7-nitrobenzofurazan