Protein Structure, Dynamics, and Aggregation in Phase Separated Droplets

蛋白质在相分离液滴中的结构、动力学和聚集 摘要 肌萎缩侧索硬化症(ALS)是一种严重而致命的疾病。近年来，人们发现 疾病进展的一个关键机制是许多肽的液-液相分离(Llps)。 和蛋白质。也有间接证据表明，LLP可以诱导蛋白质折叠/聚集成淀粉样蛋白- 就像水凝胶在许多不同的疾病中一样，包括肌萎缩侧索硬化症、2型糖尿病和阿尔茨海默病。我们 最近报道的直接原位证据表明，相分离会导致肽和蛋白质的折叠转变 来自肌萎缩侧索硬化症的蛋白质。该提案旨在以这项工作为基础，开发和应用光谱工具 原位表征蛋白质的结构、动力学和相分离液滴中的溶剂化作用，以便 确定这些折叠蛋白质和凝胶的结构和形成机制，并研究这些 变化与ALS的疾病状态有关。为了做到这一点，我们将使用二维红外 光谱(2DIR)、红外显微镜和2DIR显微镜，以探测二级结构和 水滴中的多肽和蛋白质的水合作用，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：解决问题在驾驶中起什么作用 管理有限责任合伙的力量？聚合物致密的LLP液滴中的体积拥挤能否促进蛋白质的变化 二级结构？LLP能使蛋白质折叠/聚集进入潜在的有毒淀粉样蛋白状态吗？我们会 能够为现场研究回答这些问题，这是目前其他技术不可能做到的。 这项提案中概述的战略是以建立一个研究计划为长期目标的 可以在复杂的生物物理系统中进行结构研究，使全套结构敏感 在整个细胞系统中解决问题的非线性红外光谱学中的可观察性。

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.