TAU PROTEIN AGGREGATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们正在开发一种新技术，通过监测特定蛋白质位点暴露于水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，也称为Overhauser效应，其中电子和质子之间的耦合由溶液状态分子的平移分子运动介导。通过饱和稳定的氮氧自由基的未成对电子的电子自旋共振（ESR）之一，我们能够将其部分极化转移到耦合质子，从而增加NMR信号。结合位点特异性自旋标记的蛋白质的水质子的增强信号给出了关于在特定蛋白质位点的局部溶剂动力学的信息。我们能够利用这样的表征研究tau蛋白的聚集，因为局部溶剂动力学变化显着，蛋白质位点进行结合和聚集。 以这种方式在tau蛋白的各个位点监测聚集动力学。我们的DNP衍生的动力学数据进行了解释和验证的其他几个既定的方法，如EM，荧光和浊度测量。还采用ESR信号随时间变宽的观察来获得基于自旋标记之间的tau间接近性的调节和/或不同tau种类（单体、聚集体等）的旋转动力学的变化的tau聚集的动力学数据。由于用于聚集的诱导分子（此处为肝素）的浓度不同，观察到tau聚集的动力学速率。随着肝素平均链长从3 kDa变为6 kDa再变为18 kDa，观察到聚集率降低。从几个自旋标记的突变体的动力学数据进行了比较，其中大多数突变体显示细长的纤维形态的EM是典型的淀粉样蛋白斑块，而一个突变体不形成纤维，但无定形团聚体。 我们想获得的tau纤维的图像，因为它们形成在周围的溶液条件下原位的聚集过程中，伴随和支持我们的表征tau聚集在溶液状态下的动态核极化技术研究。 我们是第一个开发基于DNP的方法来监测溶液状态下蛋白质聚集过程的团队。因此，冷冻溶液状态下tau纤维的Cryo TEM图像将不代表我们研究的关键实验数据，但将提供重要的对照实验，并将作为我们实验的补充材料。我们希望在从聚集开始后10分钟到纤维形成过程的24小时的不同聚集时间点直接观察纤维的形态和尺寸。

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.