Department of Physical and Environmental Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, ON, M1C1A4, Canada.
ACS Chem Neurosci. 2013 Aug 21;4(8):1194-203. doi: 10.1021/cn400021d. Epub 2013 May 20.
The formation of neurofibrillary tangles by hyperphosphorylated tau is a well-recognized hallmark of Alzheimer's disease. Resulting from malfunctioning protein kinases, hyperphosphorylated tau is unable to bind microtubules properly, causing it to self-associate and aggregate. The effects of tau phosphorylation on tau conformation and aggregation are still largely unexplored. The conformational analysis of tau and its hyperphosphorylated forms is usually performed by a variety of spectroscopic techniques, all of which require ample sample concentrations and/or volumes. Here we report on the use of surface based electrochemical techniques that allow for detection of conformational changes and orientation of tau protein as a function of tau phosphorylation by tyrosine and serine/threonine kinases. The electrochemical methods utilize 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) derivative as a cosubstrate and tau immobilized on gold surface to probe the role of the following protein kinases: Sarcoma related kinase (Src), Abelson tyrosine kinase (Abl), tau-tubulin kinase (TTBK), proto-oncogene tyrosine protein kinase Fyn (Fyn), and glycogen synthase kinase 3-β (Gsk-3β). The single kinase and sequential kinase-catalyzed Fc-phosphorylations modulate the electrochemical signal, pointing to the dramatic changes around the Fc group in the Fc-phosphorylated tau films. The location and orientation of the Fc-group in Fc-tau film was investigated by the surface plasmon resonance based on antiferrocene antibodies. Additional surface characterization of the Fc-tau films by time-of-flight secondary ion-mass spectrometry and X-ray photoelectron spectroscopy revealed that Fc-phosphorylations influence the tau orientation and conformation on surfaces. When Fc-phosphorylations were performed in solution, the subsequently immobilized Fc-tau exhibited similar trends. This study illustrates the validity and the utility of the labeled electrochemical approach for probing the changes in protein film properties, conformation, and orientation as a function of the enzymatically catalyzed modifications.
过度磷酸化的 tau 形成神经原纤维缠结是阿尔茨海默病的一个公认标志。由于蛋白激酶功能失调，过度磷酸化的 tau 无法正确结合微管，导致其自身缔合和聚集。tau 磷酸化对 tau 构象和聚集的影响在很大程度上仍未得到探索。tau 及其过度磷酸化形式的构象分析通常通过各种光谱技术进行，所有这些技术都需要充足的样品浓度和/或体积。在这里，我们报告了使用基于表面的电化学技术，该技术允许检测 tau 蛋白构象变化和取向，作为酪氨酸和丝氨酸/苏氨酸激酶对 tau 磷酸化的功能。电化学方法利用 5'-γ- 二茂铁腺苷三磷酸（Fc-ATP）衍生物作为共底物和固定在金表面上的 tau 来探测以下蛋白激酶的作用：肉瘤相关激酶（Src）、Abelson 酪氨酸激酶（Abl）、tau-微管蛋白激酶（TTBK）、原癌基因酪氨酸蛋白激酶 Fyn（Fyn）和糖原合成酶激酶 3-β（Gsk-3β）。单一激酶和顺序激酶催化的 Fc 磷酸化调节电化学信号，表明 Fc-磷酸化 tau 膜中 Fc 基团周围发生了剧烈变化。Fc 组在 Fc-tau 膜中的位置和取向通过基于反铁蛋白抗体的表面等离子体共振进行了研究。通过飞行时间二次离子质谱和 X 射线光电子能谱对 Fc-tau 膜进行的额外表面表征表明，Fc 磷酸化会影响 tau 在表面上的取向和构象。当在溶液中进行 Fc 磷酸化时，随后固定的 Fc-tau 表现出类似的趋势。这项研究说明了标记电化学方法在探测蛋白质膜性质、构象和取向变化方面的有效性和实用性，这些变化是酶促修饰的功能。
