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Trichloroacetic acid

三氯乙酸

Company: Sigma-Aldrich
Catalog#: 27242
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Substituted Cysteine Accessibility Method for Topology and Activity Studies of Membrane Enzymes Forming Thioester Acyl Intermediates in Bacteria
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2015-11-05
[Abstract]  The topology of membrane proteins and enzymes can be determined using various methods including reporter protein fusions and accessibility of cysteine residues to alkylating agents. Here we describe a variation of the substituted cysteine accessibility method to determine membrane topology and activity of enzymes containing an active site cysteine. Membrane topology of proteins can be predicted using different programs and the actual membrane topology can be determined by monitoring the accessibility of cysteine residues introduced in periplasmic (exposed) or cytoplasmic (not exposed) loops to alkylating agents. A two-step protocol is described where whole Escherichia coli (E. coli) cells are first treated with or without a membrane impermeable thiol reagent ... [摘要]  膜蛋白和酶的拓扑学可以使用各种方法确定,包括报告蛋白融合和半胱氨酸残基对烷化剂的可达性。在这里,我们描述了取代的半胱氨酸可接近性方法的变化,以确定膜拓扑和含有活性位点半胱氨酸的酶的活性。可以使用不同的程序预测蛋白质的膜拓扑,并且可以通过监测在周质(暴露的)或细胞质(未暴露的)环中引入的半胱氨酸残基对烷化剂的可及性来确定实际的膜拓扑。描述了两步方案,其中首先用或不用膜不可渗透的硫醇试剂(2-磺酸基乙基) - 甲烷硫代磺酸盐处理整个大肠杆菌(大肠杆菌)细胞(MTSES)并随后用烷基化试剂马来酰亚胺聚乙二醇(malPEG)标记。当半胱氨酸残基可接近MTSES并且因此暴露于周质(或可从周质接近)时,它们的游离硫醇基团与MTSES共价反应,并因此被malPEG封闭以进行烷基化。胞质或膜嵌入的半胱氨酸残基的硫醇基团不能到达MTSES,并且蛋白质可以用malPEG烷基化,导致5kDa的分子量增加。在方案的第二部分中,半胱氨酸残基的可及性用于解决形成稳定的硫酯酰基中间体的酶的酰化状态。硫酯可以被中性羟胺特异性切割,导致活性位点半胱氨酸的游离巯基,然后可以用malPEG烷基化。

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