| Substituted Cysteine Accessibility Method for Topology and Activity Studies of Membrane Enzymes Forming Thioester Acyl Intermediates in Bacteria
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Author:
Date:
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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| Dictyostelium Cultivation, Transfection, Microscopy and Fractionation
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Author:
Date:
2015-06-05
[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells and isolation of organelles or known compartments can often verify any subcellular localisation and the use of tagged proteins as bait for the immunoprecipitation of material from cell fractions can identify specific binding partners and multiprotein complexes thereby helping assign a function to the tagged protein. We have successfully applied these techniques to the Dictyostelium discoideum protein TSPOON that is part of an ancient ...
[摘要] 使用更复杂的显微镜，活细胞中荧光标记的蛋白质的实时可视化大大增加了我们对基本生理过程如细胞运动，趋化性，细胞分裂和膜运输过程中关键蛋白质动力学的了解。此外，细胞的分级和分离细胞器或已知的隔室通常可以验证任何亚细胞定位，并且使用标记的蛋白质作为诱饵用于来自细胞部分的物质的免疫沉淀可以鉴定特异性结合配偶体和多蛋白复合物，从而有助于赋予功能标记蛋白。我们已经成功地将这些技术应用于作为古代异构六聚体膜转运复合物的一部分的盘基网柄菌discoideum蛋白TSPOON（Hirst等，2013）。 ...
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