Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases.
Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluorescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluorescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA-beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead-linked library and a peptide containing the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized.
Spetzler
J C
JC
Department of Chemistry, Carlsberg Laboratory, Valby, Denmark.
Westphal
V
V
Winther
J R
JR
Meldal
M
M
D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D004220:Disulfides; D005456:Fluorescent Dyes; D019151:Peptide Library; D010455:Peptides; D003553:Cystine; D019704:Protein Disulfide-Isomerases