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

职业：核心组蛋白的折叠：核小体折叠的见解

• 批准号: 9983831
• 负责人: Lisa Gloss
• 金额: $ 48.59万
• 依托单位: Washington State University
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9983831&HistoricalAwards=false
• 关键词: CAREER; Folding; Core; Histones; Insights

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.

Gloss 9983831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。 核心组蛋白的变体形式的平衡和动力学折叠响应将用未修饰的重组组蛋白、缺少高电荷的N-末端尾部或含有从尾部消除一个或多个碱性残基的定点突变的截短的组蛋白、在N-末端尾部上具有特定乙酰化模式的组蛋白、以及含有突变取代的组蛋白，其模拟ATP依赖性核小体重塑复合物的作用，其活性是基因调控所必需的。 这些组蛋白变体的稳定性和折叠将在单独和与特定DNA片段复合的情况下进行研究，包括在完整的核小体中，这项研究将使人们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。 特别重要的是生物物理特性的调节改变所带来的组蛋白乙酰化和ATP依赖性染色质重塑复合物对组蛋白和核小体的结构和稳定性的影响。 在这些效应物存在和不存在的情况下，对核小体及其组分组蛋白进行详细的生物化学和生物物理分析，对于理解这些改变的性质以及它们在DNA包装和基因调控中所起的作用至关重要。 该教育计划包括在现有的部门课程生物物理学和蛋白质折叠的正式讲座，CD和荧光光谱及其在生化实验中的实际应用，包括但不限于，蛋白质折叠，指导研究生和本科生在PI的实验室和部门，重点是多-用学科方法解决生物学问题。