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

Site-directed covalent mnodification of bacteriorhodopsin is achieved by reacting the hydrophobic probe 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBd-CI) at neutral pH with purple membranes. The basteriorhodosin fluorescence thus produced is specific for a nucleophgilic group. the spectral properties of NBd- modified bsterorhodopsin indicate covalent interaction of the probe with thge nuclephilic ɛ-amino group of a luysine reside. Modification fo tyrosine can be exclude. As demonstrated by polypeptide fragmentation and subsequent sequence analysis, NBD binding is confined to lysine 41 within the primary structure of bacteriorhodopsin. Collisional fluorescence quendching with iodide demonstrrates that, In NBd-treated purplke membranes, the covalently bound label is not accessible in the aqueous phase. A hjysdrophobic location for the introduced fluorophor is thereby implid.