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Published on: June 7, 2018
Parameters affecting in vitro oxidation/folding of maurotoxin, a four-disulphide-bridged scorpion toxin
E di Luccio1, D O Azulay, I Regaya
1CNRS UMR 6560, Bd Pierre Dramard, 13916 Marseille Cedex 20, France.
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.
Insights
This study details the in vitro folding of synthetic Maurotoxin (MTX), a scorpion toxin. Researchers found that cellular enzymes like PPIase and PDI stereoselectively influence folding kinetics, with optimal conditions identified for efficient refolding.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Maurotoxin (MTX) is a scorpion-derived peptide toxin targeting potassium channels.
- MTX features a conserved alpha/beta scaffold stabilized by four disulfide bridges.
- Understanding MTX folding is crucial for toxin structure-function studies and therapeutic development.
Purpose of the Study:
- To investigate the in vitro oxidation and folding kinetics of synthetic L-Maurotoxin (L-sMTX).
- To develop an accurate mathematical model describing MTX folding.
- To compare the folding of L-sMTX with its stereoisomer, D-sMTX, and assess the impact of cellular enzymes.
Main Methods:
- In vitro oxidative folding of reduced L-sMTX and D-sMTX.
- Mass spectrometry (MS) analysis of folding intermediates via iodoacetamide alkylation.
- Mathematical modeling of folding kinetics, including the diffusion-collision model.
- Systematic variation of experimental parameters (temperature, pH, ionic strength, redox potential, concentration).
- Assessment of peptidylprolyl cis-trans isomerase (PPIase) and protein disulfide isomerase (PDI) effects.
Main Results:
- Folding intermediates of L-sMTX appeared sequentially, from mono- to tetra-disulfide bridged species.
- A modified mathematical model provided a better fit to the experimental folding data than the standard diffusion-collision model.
- Folding kinetics of L-sMTX and D-sMTX were largely similar across various conditions, except in the presence of stereospecific enzymes.
- Optimal folding conditions were identified: 50 mM Tris/HCl/1.4 mM EDTA, pH 7.5, with 0.5 mM PPIase and 50 units/ml PDI for 0.1 mM reduced compound.
- Significant stereoselective effects of PPIase and PDI on MTX folding were observed.
Conclusions:
- The study elucidates the complex in vitro oxidative folding pathway of synthetic Maurotoxin.
- A novel mathematical framework accurately describes MTX folding kinetics.
- Cellular enzymes PPIase and PDI exhibit potent stereoselective activity in modulating MTX folding.
- These findings offer insights into protein folding mechanisms and potential enzyme-assisted refolding strategies for scorpion toxins.
