Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins
Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins
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DOI：
10.1038/nprot.2007.275
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Kaback, H. Ronald
中科院分区：
文献类型：
--
作者：
Guan, Lan;Kaback, H. Ronald
本方案描述了一种详细的方法来研究膜蛋白的静态和动态特征，以及溶剂的可及性，利用大肠杆菌的乳糖渗透酶（LacY）作为模型。该方法依赖于使用功能性单cys突变体、亲和标签和PhosphoImager。膜渗透的放射性硫醇试剂N-[乙基-1-C-14]乙基马来酰亚胺（[C-14] NEM）用于原位检测工程单cys突变体的定点烷基化。Cys残基的溶剂可及性也通过用膜外硫醇试剂（如甲乙硫磺酸乙磺酸盐（MTSES））堵塞[C-14] NEM标记来确定。标记的蛋白用小型亲和层析纯化，并用凝胶电泳分析。凝胶干燥并暴露在磷光成像仪屏幕上1-5天，可见放射性的掺入。24h后可得到初步结果。
This protocol describes a detailed method to study the static and dynamic features of membrane proteins, as well as solvent accessibility, by utilizing the lactose permease of Escherichia coli (LacY) as a model. The method relies on the use of functional single-Cys mutants, an affinity tag and a PhosphoImager. The membrane-permeant, radioactive thiol reagent N-[ethyl-1-C-14]ethylmaleimide ([C-14] NEM) is used to detect site-directed alkylation of engineered single-Cys mutants in situ. The solvent accessibility of the Cys residues is also determined by blockage of [C-14] NEM labeling with membrane-impermeant thiol reagents such as methanethiosulfonate ethylsulfonate (MTSES). The labeled proteins are purified by mini-scale affinity chromatography and analyzed by gel electrophoresis. Gels are dried and exposed to a PhosphoImager screen for 1-5 d, and incorporation of radioactivity is visualized. Initial results can be obtained in 24 h.