Effect of Cu2+ on the oxidative folding of synthetic maurotoxin in vitro.
Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K+ channel types. It folds according to an alpha/beta scaffold, i.e., a helix connected to a two stranded beta-sheet by two disulphide bridges. In a former study, various parameters that affect the oxidation and folding of the reduced form of synthetic MTX were investigated in vitro. It was found that MTX achieves its final 3-D structure by evolving over time through a series of oxidation intermediates, from the least to the most oxidized species. MTX oxidative intermediates can be studied by iodoacetamide alkylation of free cysteine residues followed by mass spectrometry analysis. Here, we have analysed the effect of Cu2+ (0.1 to 50 mM) on the kinetics of MTX oxidative folding and found that it dramatically speeds up the formation of the four-disulphide bridged, native-like, MTX (maximal production within 30 minutes instead of > 60 hours). This catalysing effect of Cu2+ was found to be concentration-dependent, reaching a plateau at 10 mM copper ions. Cu2+ was also found to prevent the slow transition of a three disulphide-bridged MTX intermediate towards the final four disulphide-bridged product (12% of total MTX). The data are discussed in light of the potential effects of Cu2+ on MTX secondary structure formation, disulphide bridging and peptidyl prolyl cis-trans isomerization.
ERT 62 "Ingénierie des protéines", Université de la Méditerranée-Ambrilia Biopharma Inc., Faculté de Médecine Nord, Boulevard Pierre Dramard, 13916 Marseille Cédex 20, France.
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I
Regaya
N
N
Andreotti
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E
Di Luccio
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M
De Waard
J-M
JM
Sabatier
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003300:Copper / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry*; D066298:In Vitro Techniques; D007460:Iodoacetamide / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry*; D010084:Oxidation-Reduction; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012604:Scorpion Venoms / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D009498:Neurotoxins; D012604:Scorpion Venoms; C104498:maurotoxin; D003300:Copper; D007460:Iodoacetamide