使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析
Summary
We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.
Abstract
We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.
Introduction
适应性反应是细菌生存的关键。为了检测和环境的变化作出反应，细菌使用一种称为双组分信令的刺激反应系统。 1,2在典型的双组分体系中，组氨酸激酶检测同源的刺激，autophosphorylates其保守组氨酸残基，然后传送磷酸盐上的反应调节蛋白的接收机结构域的保守天冬氨酸残基。 3该事件触发的响应调节，刺激下游效应的活性的改变。 4,5-因此，细菌能够感知和适应本地环境的变化。一些双组分信号系统从这个原型偏离。在一些情况下，组氨酸激酶的感官域是一个独立的蛋白，它直接检测感知输入，并通过蛋白质 – 蛋白质相互作用修改激酶活性。 6 – 8然而，基金amental过程和系统的整体作用是相同的。双组分信令是一个普遍存在的刺激反应系统，该系统对细菌存活是必不可少的，和组氨酸激酶在信号转导中起关键作用。 9
尽管组氨酸激酶细菌生物学的重要性，他们仍然很少描述。这是由于phosphohistidine固有的不稳定性，以及缺乏用于测量自磷酸化的实用的方法。 Phosphohistidine比磷酸丝氨酸，磷酸苏和磷酸更不稳定。 10因此，通常用于分析丝氨酸/苏氨酸/酪氨酸激酶的技术是不适用的组氨酸激酶。 11 在体外测定来研究组氨酸激酶基本上都被限制在SDS-PAGE上放射自显影。 12,13在该方法中，[γ-32 p] -ATP一起温育与激酶和激酶的磷酸化是由聚合酶分析yacrylamide凝胶电泳（PAGE），随后该凝胶的放射自显影。此方法可用于监测激酶自身磷酸化，以及从所述激酶反应调节磷酸转移。然而，这种方法具有显着的缺点。基于页面的测定是低吞吐量和费时。这样的限制是不利于表征蛋白并确定其动力学参数。这是最近公布的研究组氨酸激酶的另一种方法是利用phosphohistidine抗体来检测磷酸化。 14虽然这种方法具有1- phosphohistidine和3- phosphohistidine区分的优点，这取决于用于检测的仪器，这种方法可能不提供大的动态范围或检测的高上限。因此，需要一种可用于研究这些重要的蛋白质一更快，更费力，更敏感的检测。
这里，我们描述以及demonstrate一个精心开发硝化纤维结合测定法可用于定量在体外纯化细菌组氨酸激酶的磷酸化。该测定是更高的吞吐量和耗时比基于PAGE的测定以下。该方法也利用了phosphohistidine量化，它提供了检测和大的动态范围的高上限切伦科夫辐射。该测定可用于确定用于组氨酸激酶动力学参数。
Protocol
Representative Results
Discussion
我们所描述的硝化纤维结合分析具有比以前使用的方法很多优势表征组氨酸激酶。相较于传统的SDS /基于页面的放射自显影，我们的方法是更高的吞吐量和更少耗费时间。硝酸纤维素膜更容易处理比SDS凝胶，并且不需要是固定的。丽春染色硝化棉允许而被可视化的蛋白斑点。这提供了一种简单的方法来切出每个点的闪烁计数，并确定蛋白质加载在所有点是一致的。每个点的闪烁计数提供了可以使?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由教育部通过国家需求计划（P200A100044）领域的研究生援助支持。
Materials
| Phosphoric acid | VWR | AAAA18067-AP | For quenching reactions and washing nitrocellulose |
| Tris base | RPI | T60040-5000.0 | Tris-HCl pH 8.0, for kinase reaction buffer |
| Potassium chloride | RPI | P41000-2500.0 | For kinase reaction buffer |
| Magnesium chloride | RPI | M24000-500.0 | For kinase reaction buffer |
| Glycerol | RPI | G22020-4000.0 | For kinase reaction buffer |
| 5'-ATP | Promega | E6011 | Kinase substrate |
| [γ-32P]-5'-ATP | Perkin Elmer | NEG002Z250UC | 6000 Ci/mmol |
| 96-well dot blot apparatus | Bio-rad | 1706545 | For spotting reactions |
| Nitrocellulose | Whatman | 32-10401396-PK | For spotting reactions |
| Ponceau S | Sigma aldrich | P3504-50G | For staining nitrocellulose |
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