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

平衡和停流CD和荧光(FL)光谱的生物物理技术将被用来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。不同形式的核心组蛋白的平衡和动态折叠反应将由以下几种因素决定：未修饰的重组组蛋白、缺少高电荷N-末端尾巴或包含从尾巴中消除一个或多个碱性残基的定点突变的截短组蛋白、在N-末端具有特定乙酰化模式的组蛋白，以及包含突变取代的组蛋白，这些突变替代模仿依赖于ATP的核小体重塑复合体的影响，其活性对基因调控至关重要。这些组蛋白变体的稳定性和折叠将在分离的和与特定DNA片段的复合体中进行检查，包括在完整的核小体中。这一研究将使我们从总体上更深入地了解寡聚蛋白质的折叠反应，并为核小体的组装和稳定性提供见解。尤其重要的是组蛋白乙酰化和依赖于ATP的染色质重塑复合体对组蛋白和核小体结构和稳定性所带来的调节变化的生物物理特征。在有无这些效应因子的情况下，对核小体及其组蛋白进行详细的生化和生物物理分析，对于了解这些变化的性质以及它们在DNA包装和基因调控中所起的作用是至关重要的。教育计划包括在现有的系课程中进行生物物理学和蛋白质折叠的正式讲座，开发一门以技术为导向的CD和荧光光谱学课程及其在生化实验中的实际应用，包括但不限于蛋白质折叠，在PI的实验室和系对研究生和本科生进行指导，重点是采用多学科方法解决生物问题。

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.