Protein Footprinting Coupled to Mass Spectrometry for the Study of Protein Higher Order Structure in Complex Model Systems

蛋白质足迹与质谱联用用于复杂模型系统中蛋白质高阶结构的研究

• 批准号: 10707250
• 负责人: Lisa M Jones
• 金额: $ 57.15万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707250
• 关键词: Affect; Affinity; Amino Acids; Animal Model; Binding; Biological Models; Caenorhabditis elegans; Cell Culture Techniques; Cells; Complex; Coupled; Crowding; Development; Diffusion; Environment; Higher Order Chromatin Structure; Hydroxyl Radical; In Vitro; Ligands; Mass Spectrum Analysis; Methods; Protein Conformation; Protein Footprinting; Protein Region; Proteins; Research; Site; Solvents; System; Tissues; design; human disease; in vivo; interest; macromolecule; method development; monolayer; novel therapeutics; oxidation; protein aggregation; protein folding; protein function; protein misfolding; protein protein interaction; protein structure; three-dimensional modeling

Project Summary The cellular environment is crowded with a concentration of macromolecules between 200-400 grams per liter. This crowding affects protein interactions, binding affinities, and diffusion. These conditions are not replicated in the dilute solutions used for in vitro studies. To have a full understanding of protein function, it is necessary to study proteins in complex environments that mimic the in vivo environment. However, the high concentration of macromolecules makes it difficult to perform structural studies in these systems. Owing to this, it is necessary to develop new methods to study protein structure in complex model systems. Here, we propose to further establish the protein footprinting method fast photochemical oxidation of proteins (FPOP) for studying complex model systems. FPOP utilizes hydroxyl radicals to oxidatively modify solvent accessible amino acids in proteins. The in vitro method can identify protein-ligand and protein-protein interaction sites as well as regions of protein conformation changes. My group has further expanded FPOP for studies in cells (IC-FPOP) and in vivo (IV-FPOP) in C. elegans, an animal model for human disease. We have demonstrated that IC- and IV-FPOP can oxidatively modify hundreds to thousands of proteins in these complex systems. The next step in method development is to establish their efficacy for identifying protein interactions in these model systems by studying specific applications. For the next 5 years, we plan to apply IC- and IV-FPOP to study protein folding and aggregation. The identification of protein interactions involved in misfolding and aggregation will help design new therapeutics. We also plan to extend the method into another three-dimensional model system, ex vivo tissue. This will provide structural information in a model system that more closely resembles the in vivo environment than monolayer cell culture.

项目摘要 细胞环境中聚集着大量的大分子， 每升200-400克。这种拥挤影响蛋白质相互作用，结合亲和力， 扩散这些条件在用于体外试验的稀释溶液中不重复。 问题研究为了充分了解蛋白质的功能，有必要研究 在模拟体内环境的复杂环境中的蛋白质。但高 大分子的浓度使得难以在这些化合物中进行结构研究。 系统.因此，有必要发展新的蛋白质研究方法 在复杂的模型系统中。在这里，我们建议进一步建立蛋白质 蛋白质快速光化学氧化法（FPOP） 复杂的模型系统FPOP利用羟基自由基氧化改性溶剂 蛋白质中的氨基酸。该体外方法可以鉴定蛋白质-配体， 蛋白质-蛋白质相互作用位点以及蛋白质构象变化的区域。我 该小组进一步扩展了FPOP的细胞研究（IC-FPOP）和体内研究（IV-FPOP） in C. elegans，一种人类疾病的动物模型。我们已经证明，IC-和 IV-FPOP可以氧化修饰这些复合物中的数百至数千种蛋白质 系统.方法开发的下一步是确定它们对以下疾病的疗效： 通过研究特定的应用来识别这些模型系统中的蛋白质相互作用。 在接下来的5年里，我们计划应用IC-和IV-FPOP来研究蛋白质折叠， 聚合来蛋白质相互作用的鉴定涉及错误折叠和 聚合将有助于设计新的疗法。我们还计划将该方法扩展到 另一个三维模型系统，离体组织。这将提供结构性 模型系统中的信息更接近于体内环境， 单层细胞培养