Light activates the reaction of bacteriorhodopsin aspartic acid-115 with dicyclohexylcarbodiimide.
Conditions for a light-induced reaction between the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) and bacteriorhodopsin in Triton X-100 micelles were previously reported [Renthal, R., Dawson, N., & Villarreal, L. (1981) Biochem. Biophys. Res. Commun. 101, 653-657]. We have now located the DCCD site in the bacteriorhodopsin amino acid sequence. [14C]DCCD-bacteriorhodopsin (0.67 mol/mol of bacteriorhodopsin) was cleaved with CNBr. The resulting peptides were purified by gel filtration and reverse-phase high-performance liquid chromatography (HPLC). One major 14C peptide (50%) and two minor fractions were obtained. The modified peptides were completely absent in the absence of DCCD, and 10 times less was obtained when the reaction was run in the dark. Amino acid analysis and sequence analysis showed that the major fraction contained residues 69-118. This region includes six carboxyl side chains. Quantitative sequence analysis ruled out significant amounts of DCCD at Glu-74, Asp-85, Asp-96, Asp-102, and Asp-104. The major 14C peptide was also subjected to pepsin hydrolysis. HPLC analysis of the product gave only a single major radioactive subfragment. Amino acid analysis of the peptic peptide showed that it contained residues 110-118. The only carboxyl side chain in this region is Asp-115. Thus, we conclude that Asp-115 is the major DCCD site. The light sensitivity of this reaction suggests that Asp-115 becomes more exposed or that its environment becomes more acidic during proton pumping. The DCCD reaction blue-shifts the retinal chromophore. Such a result would be expected if Asp-115 is the negative point charge predicted to be near the cyclohexene ring of retinal.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000378:metabolism* / Q000528:radiation effects; D001665:Binding Sites; D002234:Carbodiimides / Q000378:metabolism*; D002338:Carotenoids / Q000378:metabolism*; D003488:Cyanogen Bromide; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism*; D006217:Halobacterium / Q000378:metabolism*; D008027:Light; D010434:Pepsin A; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding
D000596:Amino Acids; D002234:Carbodiimides; D010446:Peptide Fragments; D001224:Aspartic Acid; D002338:Carotenoids; D001436:Bacteriorhodopsins; D004024:Dicyclohexylcarbodiimide; D010434:Pepsin A; D003488:Cyanogen Bromide