CAREER: Folding of the Core Histones: Insights into Nucleosome Folding

Gloss9983831平衡和停流CD和荧光（FL）光谱的生物物理技术将用于表征核心核小体和核小体本身的组蛋白的稳定性和折叠反应。 核心组蛋白变体形式的平衡和动力学折叠反应将由未修饰的重组组蛋白、缺乏高电荷 N 末端尾部或含有消除尾部一个或多个碱性残基的定点突变的截短组蛋白、N 末端尾部具有特定乙酰化模式的组蛋白以及含有模拟 ATP 依赖性核小体重塑复合物作用的突变取代的组蛋白决定，其活性对于基因调控至关重要。 这些组蛋白变体的稳定性和折叠将被单独检查，并与特定 DNA 片段复合，包括在完整的核小体中。这项研究将有助于更深入地了解寡聚蛋白的折叠反应，并为核小体的组装和稳定性提供见解。 特别重要的是组蛋白乙酰化和 ATP 依赖性染色质重塑复合物带来的调节改变对组蛋白和核小体结构和稳定性影响的生物物理表征。 在存在和不存在这些效应子的情况下，对核小体及其组分组蛋白进行详细的生化和生物物理分析对于了解这些改变的性质及其在 DNA 包装和基因调控中发挥的作用至关重要。 教育计划包括在现有系课程中关于生物物理学和蛋白质折叠的正式讲座、开发关于 CD 和荧光光谱及其在生化实验中的实际应用的技术导向课程，包括但不限于蛋白质折叠、在 PI 实验室和系中对研究生和本科生进行指导，重点是解决生物学问题的多学科方法。

Gloss9983831The biophysical techniques of equilibrium and stopped-flow CD and fluorescence (FL) spectroscopies will be used to characterize the stability and folding reactions of the histone proteins of the core nucleosome and the nucleosome itself. The equilibrium and kinetic folding responses of variant forms of the core histones will determined with unmodified recombinant histones, truncated histones that lack the highly charged N-terminal tails or contain site-directed mutations that eliminate one or more basic residues from the tails, histones with specific acetylation patterns on the N-terminal tails, and histones containing mutational substitutions that mimic the effects of the ATP-dependent nucleosome remodeling complexes, whose activity is essential for gene regulation. The stability and folding of these histones variants will be examined in isolation and in complex with specific DNA fragments, including in the intact nucleosome.This research will permit a deeper understanding of the folding reactions of oligomeric proteins in general and provide insights into the assembly and stability of the nucleosome. Of particular importance is the biophysical characterization of the effects of the regulatory alterations brought about by histone acetylation and ATP-dependent chromatin remodelling complexes on histone and nucleosome structure and stability. Detailed biochemical and biophysical analyses of the nucleosome and its component histones, in the presence and absence of these effectors, are essential to understanding the nature of these alterations and the role they play in DNA packaging and gene regulation. The educational plan includes formal lectures on biophysics and protein folding in existing departmental courses, development of a techniques-oriented course on CD and fluorescence spectroscopies and their practical applications to biochemical experiments, including, but not limited to, protein folding, mentoring of graduate and undergraduate students in the PI's laboratory and in the department with an emphasis on multi-disciplinary approaches to biological problems.