Topology of transmembrane proteins by scanning cysteine accessibility mutagenesis methodology
Topology of transmembrane proteins by scanning cysteine accessibility mutagenesis methodology
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DOI：
10.1016/j.ymeth.2006.08.004
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发表时间：
2007-04-01
期刊：
影响因子：
4.8
通讯作者：
Casey, Joseph R.
中科院分区：
文献类型：
--
作者：
Zhu, Quansheng;Casey, Joseph R.
质膜的整合膜蛋白从细胞内部跨越到细胞外部。完整膜蛋白的主要结构元素是它们的拓扑结构：蛋白质穿过膜的模式。拓扑学的完整描述，定义蛋白质的哪些部分面向外部和内部，对理解这些蛋白质的折叠有很大帮助。已经建立了许多方法来定义膜蛋白拓扑结构。在这里，我们提出了扫描半胱氨酸可及性诱变（SCAM）的技术。这种方法使用半胱氨酸巯基的独特化学反应性来探测膜蛋白结构。通过诱变将单个半胱氨酸残基引入靶蛋白中。使用巯基导向的试剂（膜渗透剂或非渗透剂）使这些残基化学反应的能力将每个位点定义为细胞外或细胞内，从而建立位置的拓扑结构。在蛋白质的许多位点上进行的这种分析将定义蛋白质的拓扑结构。(c)2006年爱思唯尔公司All rights reserved.
Integral membrane proteins of the plasma membrane span from the inside to the outside of the cell. The primary structural element of integral membrane proteins is their topology: the pattern in which the protein traverses the membrane. A full description of topology, defining which parts of the protein face outside versus inside, goes a long way toward understanding the folding of these proteins. Many approaches have been established to define membrane protein topology. Here, we present the technique of scanning cysteine accessibility mutagenesis (SCAM). This approach uses the unique chemical reactivity of the cysteine sulfhydryl to probe membrane protein structure. Individual cysteine residues are introduced into the target protein by mutagenesis. The ability to chemically react these residues using sulfhydryl-directed reagents (either membrane permeant or impermeant) defines each site as either extracellular or intracellular, thus establishing topology of a location. This analysis performed on many sites in the protein will define the protein's topology. (c) 2006 Elsevier Inc. All rights reserved.