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Affinity purification of cross-ß fibrils using immobilized thioflavin

使用固定化硫代黄素对交叉原纤维进行亲和纯化

基本信息

批准号：
10646061
负责人：
Wen Jiang
金额：
$ 18.43万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10646061
关键词：
3-Dimensional Actins Affinity Affinity Chromatography Alzheimer&apos s Disease American Amino Acid Sequence Amyloid Amyloid Proteins Amyloid beta-Protein Area Autopsy Binding Biological Assay Brain Buffers Cell Culture Techniques Cell model Centrifugation Cerebrospinal Fluid Characteristics Collagen Column Chromatography Congo Red Cryoelectron Microscopy Dementia Deposition Detergents Development Diagnostic Procedure Diagnostic Reagent Disease Disease Progression Drug Design Dyes Economic Burden Economics Etiology Exclusion Exhibits Feasibility Studies Ferritin Filament Fluorescence Fluorescent Probes Freezing Hour Image Immobilization In Vitro Individual Kinetics Length Life Expectancy Mass Spectrum Analysis Methods Modernization Molecular Conformation Movement Disorders Nature Neurodegenerative Disorders Nuclear Magnetic Resonance Oxides Patients Peptides Pharmaceutical Preparations Post-Translational Protein Processing Preparation Prions Procedures Property Protein Analysis Proteins Recombinant Proteins Recombinants Reporting Research Personnel Resolution Sampling Senile Plaques Severities Societies Source Specificity Surface Therapeutic Thioflavin S Time Tissue Sample Ultracentrifugation Vesicle Width Work X-Ray Crystallography alpha synuclein amyloid formation amyloid structure brain tissue cost density design experimental study feasibility testing graphene histological specimens improved in vivo interest magnetic beads meter monolayer motor disorder neuropathology preservation prevent protein aggregation protein folding protein purification protein structure reconstruction socioeconomics structural biology tau Proteins technology development tissue degeneration tool

项目摘要

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.

项目概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。截至目前，尚无可用药物可以治疗或预防这些疾病。对于合理的药物设计，了解药物的原子细节至关重要参与这些斑块的蛋白质。为了实现这一目标，研究人员利用结构生物学方法，例如电子冷冻显微镜 (cryoEM)、核磁共振和 X 射线晶体学。神经病理学家从死后患者脑组织中分离出这些斑块，并对疾病进行评估斑块的存在。然后对样品进行结构研究。自 2017 年以来，显着斑块中涉及的蛋白质分子的原子结构的数量已被报道。然而，由于产量低、蛋白质结构差异，从组织样品中分离蛋白质是一个瓶颈，以及污染物的存在。斑块中的蛋白质具有独特的排列，它们形成 β-链构象并沿螺旋轴彼此堆叠。这种安排导致一种独特的特性——它们与硫代黄素等分子结合并表现出特征性的荧光发射。这种特性使研究人员能够了解蛋白质斑块的存在并在细胞中对其进行量化模型。在这里，我们建议利用硫代黄素对神经病理学蛋白丝的特异性噬菌斑作为一种新的噬菌斑净化方法。该方法结合了硫代黄素对蛋白质丝和广泛用于纯化蛋白质的基于亲和力的纯化方法。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丝大大提高产量、减少污染物并缩短加工时间。