Altered protein-chromophore interaction in dicyclohexylcarbodiimide-modified purple membrane sheets

Abstract

Previous studies of N,N′-dicyclohexylcarbodiimide (DCCD)-modified bacteriorhodopsin (Kenthal, R. et al. (1985) Biochemistry 24, 4275–4279) used reaction conditions (detergent micelles) that are not optimal for subsequent physical studies. The present work describes new conditions for reaction of bacteriorhodopsin with DCCD in intact purple membrane sheets in the presence of 4.5% (v/v) diethylether and light. Like the detergent reaction system, the reaction is light induced, incorporates approximately 1 mol [P 1·1C]DCCD per mol bacteriorhodopsin, and results in a bleached chromophore. Peptide mapping indicates that the likely site of modification in intact membranes is identical to the site in the detergent reaction system: Asp 115. The retinal chromophore of DCCD-modified purple membrane has an absorbance maximum at 390 nm and very little induced circular dichroism. The retinal is easily extracted in hexane, yielding a 3:1 ratio of all- trans to 13- cis retinal. Borohydride reduces the retinal onto the protein within the 1–71 region of the amino acid sequence. These results suggest that Asp-115 is near the retinal binding cavity of bacteriorhodopsin. When DCCD reacts with Asp 115, retinal is displaced from its binding site.