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 细胞环境挤满了大分子之间的大分子 每升200-400克。这种拥挤会影响蛋白质相互作用，结合亲和力和 扩散。这些条件未在用于体外的稀溶液中复制 研究。要充分了解蛋白质功能，有必要研究 在模仿体内环境的复杂环境中的蛋白质。但是，高 大分子的浓度使得在这些中很难进行结构研究 系统。因此，有必要开发新方法来研究蛋白质 复杂模型系统中的结构。在这里，我们建议进一步建立蛋白质 足迹方法用于研究蛋白质（FPOP）的快速光化学氧化 复杂的模型系统。 FPOP利用羟基自由基进行氧化修饰的溶剂 蛋白质中可访问的氨基酸。体外方法可以鉴定蛋白质 - 配体，并且 蛋白质 - 蛋白质相互作用位点以及蛋白质构象的区域变化。我的 组进一步扩展了用于细胞研究（IC-FPOP）和体内研究的FPOP（IV-FPOP） 在秀丽隐杆线虫中，是人类疾病的动物模型。我们已经证明了IC和 IV-FPOP可以在这些复合物中氧化为数百种蛋白 系统。方法开发的下一步是确定其功效 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年中，我们计划应用IC和IV-FPOP来研究蛋白质折叠和 聚合。鉴定涉及错误折叠和的蛋白质相互作用 聚集将有助于设计新的治疗剂。我们还计划将方法扩展到 另一个三维模型系统，离体组织。这将提供结构性 在模型系统中的信息与体内环境更相似 单层细胞培养。