Pegylation: A method for assessing topological accessibilities in Kv1.3
Pegylation: A method for assessing topological accessibilities in Kv1.3
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DOI：
10.1021/bi0107647
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发表时间：
2001-11-06
期刊：
影响因子：
2.9
通讯作者：
Deutsch, C
中科院分区：
文献类型：
--
作者：
Lu, JL;Deutsch, C
电压门控钾通道（Kv）的每个亚基包含6个假定的跨膜片段S1-S6和胞质N-末端识别结构域T1。虽然它是公认的Kv通道是四聚体结构，蛋白质-蛋白质，蛋白质-脂质，蛋白质-水界面没有精确映射。特定氨基酸的拓扑可及性可能有助于识别这些边界残基。为此，已经使用了取代的半胱氨酸可接近性方法的变体，该方法依赖于用大SH试剂、甲氧基-聚乙二醇马来酰亚胺和凝胶位移测定对可接近的SH基团进行质量标记。全长Kv 1.3以及整合到微粒体膜中的Kv 1.3片段的聚乙二醇化允许对12个天然半胱氨酸（C1-C12）以及工程化到TI-TI界面中的半胱氨酸进行拓扑表征。工程化到T1-T1界面中的半胱氨酸具有比面向细胞溶质的半胱氨酸（即，T1结构域中的C5和C末端中的C10-C12）更低的聚乙二醇化速率。
Each subunit of a voltage-gated potassium channel (Kv) contains six putative transmembrane segments, S1-S6, and a cytosolic N-terminal recognition domain, T1. Although it is well-established that Kv channels are tetrameric structures, the protein-protein, protein-lipid, and protein-aqueous interfaces are not precisely mapped. The topological accessibility of specific amino acids may help to identify these border residues. Toward this end, a variant of the substituted-cysteine-accessibility method that relies on mass-labeling of accessible SH groups with a large SH reagent, methoxy-polyethylene glycol maleirnide, and gel shift assay has been used. Pegy1ation of full-length Kv 1.3, as well as Kv 1.3 fragments, integrated into microsomal membranes, allows topological characterization of the 12 native cysteines (C1-C12), as well as cysteines engineered into a TI-TI interface. Cysteines engineered into the TI-TI interface had lower rates of pegylation than cytosolic-facing cysteines, namely, C5 in the TI domain and C10-C12 in the C terminus.