Analyzing Protein Domain Interactions in Chemoreceptors by In Vivo PEGylation.
Analyzing Protein Domain Interactions in Chemoreceptors by In Vivo PEGylation.
复制标题
通过体内聚乙二醇化分析化学感受器中蛋白质结构域的相互作用。
DOI：
10.1007/978-1-4939-7577-8_13
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Johnson,MarkS
中科院分区：
文献类型：
--
作者：
Watts,KylieJ;Johnson,MarkS
一些蛋白质的不稳定性会阻碍体外研究。这对于膜结合的趋氧受体Aer来说是正确的，它在制备膜囊泡过程中表现出显著的蛋白质水解。通透化细胞可以很好地模拟体内条件，维持细胞内环境和相互作用结构域的几何形状。在这里，我们描述了一种优化的方法来测定渗透大肠杆菌的溶剂可及性。在这种方法中，E。用甲苯和乙醇处理含有一系列半胱氨酸替代物的共表达Aer，然后加入大型巯基反应性探针PEG-mal。在SDS-PAGE上通过聚乙二醇化蛋白与非聚乙二醇化蛋白的明显大小差异将其分离。每个半胱氨酸残基聚乙二醇化的程度然后用作溶剂可及性的量度。当绘制单cys替换库时，低可达性区域可以提示相互作用的蛋白质表面。我们成功地使用这种方法揭示了Aer PAS和HAMP结构域上不可接近的表面，然后通过二硫交联显示相互作用。
The instability of some proteins can hamper in vitro studies. This is true for the membrane-bound aerotaxis receptor, Aer, which exhibits significant proteolysis during the preparation of membrane vesicles. Permeabilized cells can closely mimic in vivo conditions, maintaining the intracellular milieu and geometry of interacting domains. Here, we describe an optimized method for determining solvent accessibility in permeabilizedEscherichia colicells. In this method,E. coliexpressing Aer with a series of cysteine replacements are treated with toluene and ethanol, after which a large sulfhydryl reactive probe, PEG-mal, is added. PEGylated protein is separated from un-PEGylated protein by its apparent size difference on SDS-PAGE. The extent to which each cysteine residue becomes PEGylated is then used as a measure of solvent accessibility. When a library of single-Cys replacements is mapped, regions of low accessibility can suggest interacting protein surfaces. We successfully used this method to reveal inaccessible surfaces on both the Aer PAS and HAMP domains that were then shown by disulfide cross-linking to interact.