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 摘要 肌萎缩侧索硬化症（ALS）是一种严重和致命的疾病。近年来，人们发现， 疾病进展的关键机制在于许多肽的液-液相分离（LLPS 和蛋白质。也有间接证据表明LLPS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疾病中的应用一样，包括ALS，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诱导肽的折叠转变， 蛋白质来源于ALS。该提案旨在以这项工作为基础，开发和应用光谱工具， 蛋白质结构、动力学和相分离液滴内溶剂化的原位表征， 确定这些折叠蛋白质和凝胶的结构和形成机制，并研究这些折叠蛋白质和凝胶是如何形成的。 这些变化与ALS的疾病状态有关。为了实现这一点，我们将使用二维红外 光谱（2D）、红外显微镜和2D显微镜，以探测二级结构的变化， 水化的肽和蛋白质液滴内，并了解基本的生物物理过程 参与蛋白质LLPS。我们要回答的关键问题是：溶剂化在驱动中起什么作用？ 管理LLPS的力量？聚合物致密LLPS液滴中的体积拥挤是否会促进蛋白质 二级结构？LLPS能驱动蛋白质折叠/聚集成潜在毒性的淀粉样蛋白状态吗？我们将 能够回答这些问题的原位研究，目前不可能与其他技术。 本提案中概述的战略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， 在非线性红外光谱学中的可观测性，以解决整个细胞系统中的问题。

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.