Photoaffinity labeling of Ras converting enzyme 1 (Rce1p) using a benzophenone-containing peptide substrate.
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.
Kyro
Kelly
K
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, United States.
Manandhar
Surya P
SP
Mullen
Daniel
D
Schmidt
Walter K
WK
Distefano
Mark D
MD
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
D001577:Benzophenones; D005456:Fluorescent Dyes; D010455:Peptides; C047723:benzophenone; D010450:Endopeptidases