Structural biology of chromatin in vitro and in cells

项目摘要 由PI Galia Debelouchina领导的实验室有以下目标：1）开发结构 生物学方法学研究复杂的和动态的生物组装体在体外，和2）的扩展， 这些方法用于细胞环境中的结构生物学研究。我们的方法 开发结合固态核磁共振（NMR）光谱与国家的最先进的 化学生物学工具，用于全面描述体外和细胞中的生物系统。我们 使用这些技术来研究染色质，一种负责细胞分裂的复杂蛋白质-DNA聚合物。 基因信息在细胞核中的包装。在未来五年，我们计划重点做好以下工作 项目：1）蛋白质翻译后修饰对染色质结构和动力学的影响。 甲基化和乙酰化等修饰对染色质的致密化有着深远的影响 聚合物和包装DNA的可及性。我们计划研究 使用固态聚合物在原子分辨率下由这些修饰赋予的染色质压缩和解压缩 核磁共振波谱法。我们希望这项研究将提供独特的见解的生物物理特性的 染色质聚合物以及它们如何转化为功能性生物输出。2)的分子基础 异染色质形成。异染色质区室与基因沉默和重复 DNA序列及其形成是细胞分化和发育的重要步骤。最近的假设 表明它们是通过液-液相分离形成的。我们计划调查 促进相分离并提供该过程的第一个分子描述。3)发展 基于NMR的细胞结构生物学工具。在这里，我们计划重点介绍一个 在细胞环境中靶向特定蛋白质并选择性地增加其NMR信号的策略 细胞背景最终，我们的目标是开发一种选择性和灵敏的NMR方法， 我们记录相关的结构数据的内源量的细胞蛋白质。这些工具将提供 这是一个前所未有的机会，可以建立细胞环境的原子分辨率图像， 蛋白质结构和动力学的变化，因为我们的压力，年龄或治疗剂处理细胞。

Project Summary The laboratory led by PI Galia Debelouchina has the following objectives: 1) The development of structural biology methodology to study complex and dynamic biological assemblies in vitro, and 2) The extension of these methodologies for structural biology investigations in the cellular environment. Our methodology development combines solid-state nuclear magnetic resonance (NMR) spectroscopy with state-of-the-art chemical biology tools for the comprehensive description of biological systems both in vitro and in cells. We use these technologies to investigate chromatin, a complex protein-DNA polymer responsible for the packaging of genetic information in the nucleus. Over the next five years, we plan to focus on the following projects: 1) The influence of protein post-translational modifications on chromatin structure and dynamics. Modifications such as methylation and acetylation have profound effects on the compaction of the chromatin polymer and the accessibility of the packaged DNA. We plan to investigate the molecular mechanisms of chromatin compaction and decompaction imparted by these modifications at atomic resolution using solid-state NMR spectroscopy. We expect that this study will provide unique insights into the biophysical properties of the chromatin polymer and how they can be translated into functional biological outputs. 2) The molecular basis of heterochromatin formation. Heterochromatin compartments are associated with gene silencing and repetitive DNA sequences, and their formation is a vital step in cell differentiation and development. Recent hypotheses indicate that they are formed through liquid-liquid phase separation. We plan to investigate the interactions that promote phase separation and to provide the first molecular description of this process. 3) Development of NMR-based tools for structural biology in cells. Here, we plan to focus on the design and implementation of a strategy to target a specific protein in the cellular milieu and to increase its NMR signals selectively over the cellular background. Ultimately, we aim to develop a selective and sensitive NMR methodology that will allow us to record relevant structural data of endogenous amounts of cellular proteins. Such tools will provide the unprecedented opportunity to build an atomic resolution picture of the cellular milieu and to investigate changes in protein structure and dynamics as we stress, age or treat cells with therapeutic agents.