Affinity purification of cross-ß fibrils using immobilized thioflavin

项目摘要 神经退行性疾病，如阿尔茨海默氏病，造成重大的社会经济负担。这些 疾病表现为脑组织中的蛋白质斑块。到今天为止，还没有可用的药物来治疗 或预防这些疾病。对于合理的药物设计，了解药物分子的原子细节是至关重要的。 与这些斑块有关的蛋白质。为了实现这一目标，研究人员利用结构生物学方法，如 电子低温显微镜（cryoEM）、核磁共振和X射线晶体学。 神经病理学家从死后病人的脑组织中分离出这些斑块， 斑块的存在。然后对样品进行结构研究。自2017年以来， 已经报道了斑块中涉及的蛋白质分子的原子结构的数量。然而，在这方面， 从组织样品中分离蛋白质是一个瓶颈，因为产量低，蛋白质结构的差异， 以及污染物的存在。斑块中的蛋白质有一种独特的排列方式， β-链构象并沿螺旋轴沿着彼此堆叠。这一安排引起了 一种独特的性质-它们与硫磺素等分子结合，并显示出特征性的荧光发射。 这种性质使研究人员能够知道蛋白质斑块的存在，并在细胞中对其进行定量。 模型在这里，我们建议利用硫磺素的特异性，以蛋白丝的神经病理 噬菌斑作为一种新的噬菌斑纯化方法。该方法将硫磺素的特异性与 蛋白丝和广泛用于纯化蛋白质的基于亲和性的纯化方法。我们提出 我们的方法将使研究人员能够从脑组织中提取和分离蛋白质丝， 产量大大提高，污染物更少，加工时间更短。

Project Summary Neurodegenerative diseases such as Alzheimer’s disease cause a significant socioeconomic burden. These diseases manifest as protein plaques in the brain tissues. As of today, there are no available drugs to treat or prevent these diseases. For rational drug design, it is essential to understand the atomic details of the proteins involved in these plaques. To achieve this, researchers utilize structural biology methods such as electron cryomicroscopy (cryoEM), nuclear magnetic resonance, and X-ray crystallography. Neuropathologists isolate these plaques from post-mortem patient brain tissues and assess the disease for the presence of plaques. Then the samples are subjected to structural studies. Since 2017, a significant number of atomic structures of the protein molecules involved in the plaques have been reported. However, isolation of proteins from tissue samples is a bottleneck due to low yield, differences in the protein structures, and the presence of contaminants. The proteins in the plaques have a distinct arrangement in that they form a β-strand conformation and stack on top of each other along a helical axis. This arrangement gives rise to a unique property – they bind to molecules such as thioflavin and exhibit characteristic fluorescence emission. Such a property enables researchers to know the presence of protein plaques and quantify them in cellular models. Here, we propose to utilize the specificity of thioflavin to protein filaments of neuropathological plaques as a new method of plaque purification. The method combines the specificity of thioflavin to the protein filaments and affinity-based purification methods that are widely used to purify proteins. We propose that our method would enable researchers to extract and isolate protein filaments from brain tissues with much improved yields, fewer contaminants, and shorter processing times.