Patching Simon G, Edara Shalini, Ma Pikyee, Nakayama Jiro, Hussain Rohanah, Siligardi Giuliano, Phillips-Jones Mary K
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds. L32 9JT, UK.
Biochim Biophys Acta. 2012 Jul;1818(7):1595-602. doi: 10.1016/j.bbamem.2012.02.015.
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 kd value of 2 microM indicative of relatively loose binding ofgelatinase 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 kd 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.
FsrC是粪肠球菌医院获得性感染群体感应中Fsr双组分信号转导系统的膜结合组氨酸激酶成分。本文使用同步辐射圆二色光谱研究溶解在去污剂胶束中的完整纯化蛋白。首先通过将稳定化蛋白长时间暴露于远紫外辐射来确定和测试去污剂中FsrC稳定性所需的条件。使用稳定化的纯化蛋白，远紫外同步辐射圆二色光谱显示FsrC含有61%的α-螺旋，并且相对耐热，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℃时至少保留57%的二级结构完整性。虽然群体感应信息素配体GBAP的结合没有显著影响FsrC的二级结构，但近紫外光谱显示酪氨酸和色氨酸残基区域的三级结构受到显著影响。滴定实验显示计算出的kd值为2μM，表明明胶酶生物合成激活信息素与FsrC的结合相对松散。虽然同步辐射圆二色光谱以前已应用于膜蛋白，但据我们所知，这是首次报道用其测定完整膜蛋白的kd值。基于我们的研究结果，我们认为同步辐射圆二色光谱将是一种用于表征其他膜传感器激酶以及一般其他膜蛋白配体结合的有价值技术。它还为下游结构方法（如结晶和核磁共振实验）之前在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，特别是在使用较低去污剂浓度时。
