Interactions of the intact FsrC membrane histidine kinase with its pheromone ligand GBAP revealed through synchrotron radiation circular dichroism
Interactions of the intact FsrC membrane histidine kinase with its pheromone ligand GBAP revealed through synchrotron radiation circular dichroism
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DOI：
10.1016/j.bbamem.2012.02.015
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发表时间：
2012-07-01
影响因子：
3.4
通讯作者：
Phillips-Jones, Mary K.
中科院分区：
文献类型：
--
作者：
Patching, Simon G.;Edara, Shalini;Phillips-Jones, Mary K.
FsrC 是 Fsr 双组分信号转导系统的膜结合组氨酸激酶组分，参与医院获得性感染病原体粪肠球菌的群体感应。这里使用同步辐射圆二色光谱来研究溶解在洗涤剂胶束中的完整纯化蛋白质。首先通过将稳定的蛋白质长时间暴露于远紫外线辐射来确定和测试洗涤剂中 FsrC 稳定性所需的条件。使用稳定的纯化蛋白，远紫外同步辐射圆二色性表明 FsrC 具有 61% 的 α 螺旋，并且具有相对热稳定性，在存在或不存在明胶酶生物合成激活信息素 (GBAP) 的情况下，在 90 摄氏度下保留至少 57% 的二级结构完整性。虽然群体信息素配体 GBAP 的结合不会显着影响 FsrC 二级结构，但近紫外光谱显示 Tyr 和 Trp 残基区域的三级结构受到显着影响。滴定实验显示计算出的 k(d) 值为 2 μM，表明明胶酶生物合成激活信息素与 FsrC 的结合相对松散。尽管同步加速器辐射圆二色性的使用以前已应用于膜蛋白，但据我们所知，这是首次报告使用它来确定完整膜蛋白的 k(d) 值。根据我们的发现，我们认为同步辐射圆二色性将是一种有价值的技术，用于表征其他膜传感器激酶和其他膜蛋白的配体结合。它进一步为下游结构方法（例如结晶和核磁共振实验）之前的一系列去污剂条件下的膜蛋白稳定性提供了有价值的筛选工具，特别是当使用较低的去污剂浓度时。皇冠版权所有 (C) 2012 由 Elsevier B.V. 出版。保留所有权利。
FsrC is the membrane-bound histidine kinase component of the Fsr two-component signal transduction system involved in quorum sensing in the hospital-acquired infection agent Enterococcus faecalis. Synchrotron radiation circular dichroism spectroscopy was used here to study the intact purified protein solubilised in detergent micelles. Conditions required for FsrC stability in detergent were firstly determined and tested by prolonged exposure of stabilised protein to far-ultraviolet radiation. Using stabilised purified protein, far-ultraviolet synchrotron radiation circular dichroism revealed that FsrC is 61% alpha-helical and that it is relatively thermostable, retaining at least 57% secondary structural integrity at 90 degrees C in the presence or absence of gelatinase biosynthesis-activating pheromone (GBAP). Whilst binding of the quorum pheromone ligand GBAP did not significantly affect FsrC secondary structure, near-ultraviolet spectra revealed that the tertiary structure in the regions of the Tyr and Trp residues was significantly affected. Titration experiments revealed a calculated k(d) value of 2 mu M indicative of relatively loose binding of gelatinase biosynthesis-activating pheromone to FsrC. Although use of synchrotron radiation circular dichroism has been applied to membrane proteins previously, to our knowledge this is the first report of its use to determine a k(d) value for an intact membrane protein. Based on our findings, we suggest that synchrotron radiation circular dichroism will be a valuable technique for characterising ligand binding by other membrane sensor kinases and indeed other membrane proteins in general. It further provides a valuable screening tool for membrane protein stability under a range of detergent conditions prior to downstream structural methods such as crystallisation and NMR experiments particularly when lower detergent concentrations are used. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.