Site‐Directed Fluorogenic Modification of Bacteriophodopsin by 7‐Chloro‐4‐nitrobenz‐2‐oxa‐1,3‐diazole

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Abstract
Site-directed covalent modification of bacteriorhodopsin [from Halobacterium halobium] 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 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 implied.