di Luccio E, Azulay D O, Regaya I, Fajloun Z, Sandoz G, Mansuelle P, Kharrat R, Fathallah M, Carrega L, Estève E, Rochat H, De Waard M, Sabatier J M
CNRS UMR 6560, Bd Pierre Dramard, 13916 Marseille Cedex 20, France.
Biochem J. 2001 Sep 15;358(Pt 3):681-92. doi: 10.1042/0264-6021:3580681.
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.
毛罗毒素（MTX）是一种由四个二硫键交联的34肽蝎毒素，作用于多种钾离子通道亚型。MTX采用C1-C5、C2-C6、C3-C4和C7-C8类型的二硫键结构，并根据其他已知蝎毒素报道的常见α/β支架进行折叠。在此，我们研究了还原型合成L-MTX（L-sMTX，其中L-MTX仅含L-氨基酸残基）的体外氧化/折叠过程及动力学。在还原型L-sMTX的氧化/折叠过程中，氧化中间体通过游离半胱氨酸残基的碘乙酰胺烷基化被阻断，并通过质谱分析。L-sMTX中间体随时间依次出现，从氧化程度最低的（具有一个二硫键的中间体）到氧化程度最高的物种（天然样、具有四个二硫键的L-sMTX）。扩散-碰撞模型的数学公式不足以准确描述L-sMTX的氧化/折叠动力学，但我们已制定了一个更符合实验数据的衍生数学描述。利用这个数学描述，我们首次比较了L-sMTX与其立体异构体D-sMTX（仅含D-氨基酸残基）的氧化/折叠过程。对几个可能影响氧化/折叠过程的实验参数进行了进一步研究；这些参数包括温度、pH值、离子强度、氧化还原电位和还原毒素的浓度。我们还评估了一些细胞酶，肽基脯氨酰顺反异构酶（PPIase）和蛋白质二硫键异构酶（PDI）对还原型L-sMTX和D-sMTX折叠途径的影响。除了使用立体特异性酶的情况外，所有测试参数均影响sMTX的氧化折叠，且该过程的动力学对于L-sMTX和D-sMTX无法区分。发现最有效的条件是：50 mM Tris/HCl/1.4 mM EDTA，pH 7.5，添加0.5 mM PPIase和50单位/ml PDI用于0.1 mM还原化合物。这些数据首次报道了细胞酶对蝎毒素氧化/折叠的强效立体选择性作用。
