Functional reconstitution of Staphylococcus aureus truncated AgrC histidine kinase in a model membrane system.
Functional reconstitution of Staphylococcus aureus truncated AgrC histidine kinase in a model membrane system.
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模型膜系统中金黄色葡萄球菌截短 AgrC 组氨酸激酶的功能重建
DOI：
10.1371/journal.pone.0080400
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Fan S
中科院分区：
文献类型：
--
作者：
Wang L;Quan C;Liu B;Wang J;Xiong W;Zhao P;Fan S
膜整合蛋白AgrC是组氨酸激酶，其传感器结构域与自诱导肽相互作用，导致一系列下游反应。在本研究中，使用细菌系统产生截短的AgrCTM 5 -6C和具有GFP作为报告基因的AgrCTM 5 -6C-GFP。通过去污剂介导的方法将纯化的AgrCTM 5 -6C和AgrCTM 5 -6C-GFP重构到脂质体中。为了实现高产率的蛋白质掺入，我们研究了不同去污剂对蛋白质重构效率的影响。在完全脂质体溶解过程中，发现N，N-二甲基十二烷基胺N-氧化物的掺入量最高，产率为85± 5%。蛋白质AgrCTM 5 -6C的COOH-末端几乎完全朝向囊泡的内部。与洗涤剂胶束中的AgrCTM 5 -6C相比，蛋白脂质体中的AgrCTM 5 - 6C表现出约6倍的组成型活性增加。使用动态光散射、荧光显微镜和透射电子显微镜表征AgrCTM 5 -6C或AgrCTM 5 -6C-GFP的重建。在此基础上，确定了蛋白质掺入的最佳条件。这些发现有助于研究膜蛋白的结构和功能，在体外使用重建系统。
The integral membrane protein AgrC is a histidine kinase whose sensor domains interact with an autoinducing peptide, resulting in a series of downstream responses. In this study, truncated AgrCTM5-6C and AgrCTM5-6C-GFP with GFP as a reporter gene were produced using a bacterial system. Purified AgrCTM5-6C and AgrCTM5-6C-GFP were reconstituted into liposomes by a detergent-mediated method. To achieve high-yield protein incorporation, we investigated the effect of different detergents on protein reconstitution efficiency. The highest incorporation was found with N,N-dimethyldode-cylamine N-oxide during complete liposome solubilization, which resulted in a yield of 85±5%. The COOH-terminus of the protein AgrCTM5-6C was almost exclusively oriented towards the inside of the vesicles. AgrCTM5-6C in proteoliposomes exhibited approximately a 6-fold increase in constitutive activity compared with AgrCTM5-6C in detergent micelles. The reconstitution of AgrCTM5-6C or AgrCTM5-6C-GFP was characterized using dynamic light scattering, fluorescence microscopy, and transmission electron microscopy. Based on the results, the optimal conditions for protein incorporation were defined. These findings contribute to the study of membrane protein structure and function in vitro using a reconstitution system.
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影响因子：
3.6
作者：
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通讯作者：
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