Affinity purification of cross-ß fibrils using immobilized thioflavin

项目摘要 阿尔茨海默病等神经退行性疾病造成了巨大的社会经济负担。这些 疾病表现为脑组织中的蛋白质斑块。到今天为止，还没有可以治疗的药物。 或预防这些疾病。对于合理的药物设计，了解原子细节是必不可少的 参与这些斑块的蛋白质。为了实现这一点，研究人员利用结构生物学方法，如 电子冷冻显微镜(CryoEM)、核磁共振和X射线结晶学。 神经病理学家从死后病人的脑组织中分离出这些斑块，并评估疾病 斑块的存在。然后对样品进行结构研究。自2017年以来，显著的 斑块中涉及的蛋白质分子的原子结构的数量已经被报道。然而， 从组织样本中分离蛋白质是一个瓶颈，因为产量低，蛋白质结构不同， 以及污染物的存在。斑块中的蛋白质有不同的排列，因为它们形成了 一种β链构象，沿着螺旋轴堆叠在一起。这一安排产生了 一种独特的性质-它们与硫代黄素等分子结合，并显示出独特的荧光发射。 这种特性使研究人员能够知道蛋白质斑块的存在，并在细胞中对它们进行量化 模特们。在此，我们建议利用硫代黄素对神经病理蛋白细丝的特异性。 斑块作为一种新的斑块纯化方法。该方法结合了硫代黄素对 蛋白质长丝和亲和层析纯化方法被广泛用于蛋白质的纯化。我们建议 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细丝 大大提高了产量，减少了污染物，缩短了处理时间。

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.