title: Photoaffinity labeling of Ras converting enzyme 1 (Rce1p) using a benzophenone-containing peptide substrate.
abstract: Isoprenylation is a post-translational modification that increases protein hydrophobicity and helps target certain proteins to membranes. Ras converting enzyme 1 (Rce1p) is an endoprotease that catalyzes the removal of a three residue fragment from the C-terminus of isoprenylated proteins. To obtain structural information about this membrane protein, photoaffinity labeling agents are being prepared and employed. Here, we describe the synthesis of a benzophenone-containing peptide substrate analogue for Rce1p. Using a continuous spectrofluorometric assay, this peptide was shown to be a substrate for Rce1p. Mass spectrometry was performed to confirm the site of cleavage and structure of the processed probe. Photolysis of the biotinylated compound in the presence of membranes containing Rce1p followed by streptavidin pull-down and Western blot analysis indicated that Rce1p had been labeled by the probe. Photolysis in the presence of both the biotinylated, benzophenone-containing probe and a farnesylated peptide competitor reduced the extent of labeling, suggesting that labeling is occurring in the active site.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, United States.
authors.forename: Kelly
authors.initials: K
authors.lastname: Kyro
authors.forename: Surya P
authors.initials: SP
authors.lastname: Manandhar
authors.forename: Daniel
authors.initials: D
authors.lastname: Mullen
authors.forename: Walter K
authors.initials: WK
authors.lastname: Schmidt
authors.forename: Mark D
authors.initials: MD
authors.lastname: Distefano
mesh_terms: D000595:Amino Acid Sequence; D001577:Benzophenones / Q000737:chemistry*; D010450:Endopeptidases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D017368:Protein Prenylation; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D001577:Benzophenones; D005456:Fluorescent Dyes; D010455:Peptides; C047723:benzophenone; D010450:Endopeptidases