Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

TY - JOUR

T1 - Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

AU - Spetzler, J C

AU - Westphal, V

AU - Winther, Jakob R.

AU - Meldal, M

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

SN - 1075-2617

VL - 4

SP - 128

EP - 137

JO - Journal of Peptide Science

JF - Journal of Peptide Science

IS - 2

ER -