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Title: 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. The authors have now located the DCCD site in the bacteriorhodopsin amino acid sequence. ( UC)DCCD-bacteriorhodopsin was cleaved with CNBr. The resulting peptides were purified by gel filtration and reverse-phase high-performance liquid chromatography (HPLC). One major UC peptide (50%) and two minor fractions were obtained. Amino acid analysis and sequence analysis showed that the major fraction contained residues 69-118. This region includes six carboxyl side chains. The major UC 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, the authors 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.
Authors:
; ; ; ;
Publication Date:
OSTI Identifier:
6045375
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (United States); Journal Volume: 16
Research Org:
Univ. of Texas, San Antonio
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ASPARTIC ACID; CHEMICAL REACTIONS; CARBON 14 COMPOUNDS; AMINO ACID SEQUENCE; IMIDES; RHODOPSIN; BACTERIA; CAROTENOIDS; LIQUID COLUMN CHROMATOGRAPHY; PEPSIN; RETINA; ACID PROTEINASES; AMINO ACIDS; BODY; BODY AREAS; CARBOXYLIC ACIDS; CHROMATOGRAPHY; ENZYMES; EYES; FACE; HEAD; HYDROCARBONS; HYDROLASES; LABELLED COMPOUNDS; MICROORGANISMS; MOLECULAR STRUCTURE; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANS; PEPTIDE HYDROLASES; PIGMENTS; PROTEINS; SENSE ORGANS; SEPARATION PROCESSES; TERPENES 550201* -- Biochemistry-- Tracer Techniques