Protein Structure, Dynamics, and Aggregation in Phase Separated Droplets

相分离液滴中的蛋白质结构、动力学和聚集

• 批准号: 10713121
• 负责人: Arnaldo L Serrano
• 金额: $ 32.86万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10713121
• 关键词: Address; Alzheimer' s Disease; Amyloid; Amyotrophic Lateral Sclerosis; Biological; Biophysical Process; Biophysics; Cells; Complex; Crowding; Disease; Disease Progression; Gel; Goals; Hydration status; Hydrogels; Image; In Situ; Liquid substance; Membrane; Microscopy; Non-Insulin-Dependent Diabetes Mellitus; Organelles; Peptides; Phase; Polymers; Process; Proteins; Reporting; Research; Role; Secondary Protein Structure; Spectrum Analysis; Structure; System; Techniques; Work; design; driving force; infrared microscopy; infrared spectroscopy; programs; protein folding; protein structure; temporal measurement; tool; two-dimensional

Protein Structure, Dynamics, and Aggregation in Phase Separated Droplets Abstract Amyotrophic lateral sclerosis (ALS) is a severe and deadly disease. In recent years, it has been discovered that a key mechanism of disease progression lies in liquid-liquid phase separation (LLPS) of a number of peptides and proteins. Indirect evidence has also emerged that LLPS can induce protein folding/aggregation into amyloid- like hydrogels in a number of different diseases, including ALS, type-2 diabetes, and Alzheimer’s disease. We recently reported direct in-situ evidence that phase separation induces a folding transition for peptide and proteins derived from ALS. This proposal aims to build on that work to develop and apply spectroscopic tools for in-situ characterization of protein structure, dynamics, and solvation within phase-separated droplets, in order to identify the structure and mechanism of formation of these folded proteins and gels, and to study how these changes relate to the disease state of ALS. To accomplish this, we will use two-dimensional infrared spectroscopy (2DIR), infrared microscopy, and 2DIR microscopy, to probe changes in secondary structure and hydration of peptides and proteins within droplets, and understand the fundamental biophysical processes involved in protein LLPS. Key questions that we aim to answer are: What role does solvation serve in the driving forces governing LLPS? Can volumetric crowding in polymer dense LLPS droplets promote changes in protein secondary structure? Can LLPS drive protein folding/aggregation into potentially toxic amyloid states? We will be able to answer these questions for in-situ studies, something currently not possible with other techniques. The strategy outlined in this proposal is designed with the long-term goal of building a research program that can perform structural studies in complex biophysical systems, turning the full suite of structure sensitive observables in nonlinear IR spectroscopy towards addressing questions in whole cell systems.

蛋白质结构，动力学和相分离的液滴中的聚集 抽象的 肌萎缩性侧索硬化症（ALS）是一种严重且致命的疾病。近年来，已经发现 疾病进展的关键机制在于许多肽的液态液相分离（LLP） 和蛋白质。间接证据还表明，LLP可以诱导蛋白质折叠/聚集到淀粉样蛋白 就像许多不同疾病中的水凝胶一样，包括ALS，2型糖尿病和阿尔茨海默氏病。我们 最近报道了直接的原位证据，表明相分离会影响胡椒和 源自ALS的蛋白质。该建议旨在以这项工作为基础开发和应用光谱工具 蛋白质结构，动力学和溶液的原位表征在相位分离的液滴中，以便为了 确定这些折叠蛋白和凝胶形成的结构和机制，并研究这些蛋白质 变化与ALS的疾病状态有关。为此，我们将使用二维红外线 光谱法（2DIR），红外显微镜和2DIR显微镜，以探测二次结构和 水滴中的肽和蛋白质的水合，并了解基本的生物物理过程 参与蛋白质LLP。我们要回答的关键问题是：解决方案在驾驶中有什么作用 执政LLP的部队？聚合物密集的LLP液滴中的体积拥挤可以促进蛋白质的变化 次要结构？ LLP可以将蛋白质折叠/聚集驱动到潜在的有毒淀粉样蛋白状态吗？我们将 能够为原位研究回答这些问题，其他技术目前无法做到这一点。 该提案中概述的策略的设计旨在建立一个研究计划的长期目标 可以在复杂的生物物理系统中进行结构研究，使整个结构敏感 非线性红外光谱中的可观察物，用于解决整个细胞系统中的问题。