title: Parameters affecting in vitro oxidation/folding of maurotoxin, a four-disulphide-bridged scorpion toxin.
abstract: Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K(+) channel subtypes. MTX adopts a disulphide bridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, and folds according to the common alpha/beta scaffold reported for other known scorpion toxins. Here we have investigated the process and kinetics of the in vitro oxidation/folding of reduced synthetic L-MTX (L-sMTX, where L-MTX contains only L-amino acid residues). During the oxidation/folding of reduced L-sMTX, the oxidation intermediates were blocked by iodoacetamide alkylation of free cysteine residues, and analysed by MS. The L-sMTX intermediates appeared sequentially over time from the least (intermediates with one disulphide bridge) to the most oxidized species (native-like, four-disulphide-bridged L-sMTX). The mathematical formulation of the diffusion-collision model being inadequate to accurately describe the kinetics of oxidation/folding of L-sMTX, we have formulated a derived mathematical description that better fits the experimental data. Using this mathematical description, we have compared for the first time the oxidation/folding of L-sMTX with that of D-sMTX, its stereoisomer that contains only D-amino acid residues. Several experimental parameters, likely to affect the oxidation/folding process, were studied further; these included temperature, pH, ionic strength, redox potential and concentration of reduced toxin. We also assessed the effects of some cellular enzymes, peptidylprolyl cis-trans isomerase (PPIase) and protein disulphide isomerase (PDI), on the folding pathways of reduced L-sMTX and D-sMTX. All the parameters tested affect the oxidative folding of sMTX, and the kinetics of this process were indistinguishable for L-sMTX and D-sMTX, except when stereospecific enzymes were used. The most efficient conditions were found to be: 50 mM Tris/HCl/1.4 mM EDTA, pH 7.5, supplemented by 0.5 mM PPIase and 50 units/ml PDI for 0.1 mM reduced compound. These data represent the first report of potent stereoselective effects of cellular enzymes on the oxidation/folding of a scorpion toxin.
authors.affiliation: CNRS UMR 6560, Bd Pierre Dramard, 13916 Marseille Cedex 20, France.
authors.forename: E
authors.initials: E
authors.lastname: di Luccio
authors.forename: D O
authors.initials: DO
authors.lastname: Azulay
authors.forename: I
authors.initials: I
authors.lastname: Regaya
authors.forename: Z
authors.initials: Z
authors.lastname: Fajloun
authors.forename: G
authors.initials: G
authors.lastname: Sandoz
authors.forename: P
authors.initials: P
authors.lastname: Mansuelle
authors.forename: R
authors.initials: R
authors.lastname: Kharrat
authors.forename: M
authors.initials: M
authors.lastname: Fathallah
authors.forename: L
authors.initials: L
authors.lastname: Carrega
authors.forename: E
authors.initials: E
authors.lastname: Estève
authors.forename: H
authors.initials: H
authors.lastname: Rochat
authors.forename: M
authors.initials: M
authors.lastname: De Waard
authors.forename: J M
authors.initials: JM
authors.lastname: Sabatier
mesh_terms: D000478:Alkylation; D004220:Disulfides; D006801:Humans; D007202:Indicators and Reagents; D007460:Iodoacetamide; D007700:Kinetics; D008962:Models, Theoretical; D009498:Neurotoxins / Q000737:chemistry; D010084:Oxidation-Reduction; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D012604:Scorpion Venoms / Q000737:chemistry* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004220:Disulfides; D007202:Indicators and Reagents; D009498:Neurotoxins; D011994:Recombinant Proteins; D012604:Scorpion Venoms; C104498:maurotoxin; D019696:Peptidylprolyl Isomerase; D019704:Protein Disulfide-Isomerases; D007460:Iodoacetamide