TAU PROTEIN AGGREGATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们正在开发一种新的技术，通过监测特定蛋白质部位暴露在水中的程度来分析蛋白质聚集。这种方法依赖于溶液的动态核极化(DNP)原理，也被称为Overhauser效应，其中电子和质子之间的耦合是通过溶液状态分子的平移分子运动来调节的。通过饱和稳定的氮氧化物自由基未配对电子的一个电子自旋共振(ESR)，我们能够将其部分极化转移到一个耦合的质子上，从而导致核磁共振信号的增加。水质子的增强信号与蛋白质特定位置的自旋标记相结合，提供了特定蛋白质位置上局部溶剂动力学的信息。我们能够利用这种特征来研究tau蛋白的聚集，因为在蛋白质位点发生结合和聚集的地方，局部溶剂动力学发生了戏剧性的变化。以这种方式监测了tau蛋白不同部位的聚集动力学。我们的DNP得到的动力学数据被其他几种成熟的方法解释和验证，如EM、荧光和浊度测量。还通过观察ESR信号随时间的加宽来获得基于自旋标记之间的tau间邻近性的调制和/或不同tau物种(单体、聚集体等)的旋转动力学变化的tau聚集的动力学数据。观察到tau聚集的动力学速率是由于用于聚集的诱导剂分子的不同浓度，这里是肝素。随着肝素平均链长从3 kDa变为6 kDa到18 kDa，聚集率降低。比较了几个自旋标记突变体的动力学数据，其中大多数突变体通过EM显示了典型的淀粉样斑块的细长纤维形态，而一个突变体不形成纤维而是无定形的聚集物。 我们希望在原位获得环境溶液条件下tau纤维在聚集过程中形成的图像，以伴随和支持我们用动态核极化技术研究的溶液状态tau聚集的表征。我们是第一个开发基于DNP的方法来监控溶液状态下的蛋白质聚集过程的小组。因此，冷冻溶液中tau纤维的低温电子显微镜图像并不是我们研究的关键实验数据，而是一个重要的对照实验，并将作为我们实验的补充材料。我们想要直接观察纤维在聚集开始后10分钟到纤维形成过程长达24小时的不同时间点的形态和尺寸。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are developing a new technique for the analysis of protein aggregation by monitoring the extent to which specific protein sites are exposed to water. This approach relies on the principle of dynamic nuclear polarization (DNP) of solutions, also known as the Overhauser effect, where the coupling between electron and proton is mediated by translational molecular motion of solution state molecules. By saturating one of the electron spin resonances (ESR) of unpaired electrons of stable nitroxide radicals we are able to transfer part of its polarization to a coupled proton resulting in an increased NMR signal. Enhanced signal of water protons in combination with site-specific spin labeling of proteins gives information about the local solvent dynamics at specific protein sites. We were able to utilize such characterization in studying aggregation of tau proteins because local solvent dynamics changes dramatically where protein sites undergo binding and aggregation. The aggregation kinetics was monitored in this fashion at various sites of the tau protein. Our DNP-derived kinetic data was interpreted and verified by several other established methods such as EM, fluorescence and turbidity measurements. Observation of broadening of ESR signal over time was also employed to obtain kinetic data of tau aggregation based on modulation of inter-tau proximity between spin labels and/or change of rotational dynamics of the different tau species (monomer, aggregates etc.). Kinetic rates of tau aggregation were observed due to varying concentration of inducer molecules for aggregation, here heparin. Decrease in aggregation rates was noted with change in average chain length of heparin from 3 to 6 to 18 kDa. Kinetic data from several spin-labeled mutants were compared, in which most mutants reveal elongated fiber morphology by EM that is typical for amyloid plaques, while one mutant does not form fibers but amorphous agglomerates. We would like to obtain images of the tau fibers as they form in an aggregation process under ambient solution condition in situ, to accompany and support our characterization of tau aggregation in the solution state studied by dynamic nuclear polarization technique. We are the first group to develop a DNP based approach to monitor protein aggregation processes in solution state. Therefore, the Cryo TEM images of tau fibers in a frozen solution state will not represent key experimental data for our study, but will present an important control experiment and would serve as a supplemental material for our experiments. We would like to directly observe the morphology and size of the fibers at various time points of aggregation from 10 minutes after the start of aggregation to up to 24 hours of the fiber formation process.