Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases
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Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases. / Spetzler, Jane C.; Westphal, Vibeke; Winther, Jakob R.; Meldal, Morten.
I: Journal of Peptide Science, Bind 4, Nr. 2, 1998, s. 128-137.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases
AU - Spetzler, Jane C.
AU - Westphal, Vibeke
AU - Winther, Jakob R.
AU - Meldal, Morten
PY - 1998
Y1 - 1998
N2 - 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.
AB - 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.
KW - Cystine
KW - Disulfides
KW - Fluorescent Dyes
KW - Peptide Library
KW - Peptides
KW - Protein Disulfide-Isomerases
KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
KW - Substrate Specificity
U2 - 10.1002/(SICI)1099-1387(199804)4:2<128::AID-PSC137>3.0.CO;2-E
DO - 10.1002/(SICI)1099-1387(199804)4:2<128::AID-PSC137>3.0.CO;2-E
M3 - Journal article
C2 - 9620617
VL - 4
SP - 128
EP - 137
JO - Journal of Peptide Science
JF - Journal of Peptide Science
SN - 1075-2617
IS - 2
ER -
ID: 43974057