Probing Conformational Transition Pathways of Protein-DNA Complexes by Polarization-Modulated Fourier Imaging Correlation Spectroscopy (PM-FICS)

通过偏振调制傅立叶成像相关光谱 (PM-FICS) 探测蛋白质-DNA 复合物的构象转变途径

• 批准号: 1105272
• 负责人: Andrew Marcus
• 金额: $ 47万
• 依托单位: University of Oregon Eugene
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1105272&HistoricalAwards=false
• 关键词: Probing; Conformational; Transition; Pathways; Protein

This award in the Chemistry of Life Processes (CLP) program supports work by Professor Andrew H. Marcus at the University of Oregon to carry out fundamental studies on the molecular mechanisms of DNA polymerase and helicase, two protein-DNA complexes that are essential elements of the biological processes that enact and regulate gene expression. Pairs of fluorescent nucleic acid base analogues are substituted for native nucleotides at the forks and junctions of double stranded and primer-template DNA. Polarized fluorescence fluctuation measurements of the naked DNA constructs will reveal information about the relative dipole angle of the base analogue sites, and their relative motions on microsecond to sub-second time scales. Subsequent experiments will be carried out on protein-DNA complexes to directly probe the mechanics of the protein enzymatic sites during key steps of the replication process. These studies will provide detailed information about the transition state pathways of DNA synthesis, and investigate the possible existence of intermediate states involved in replication fidelity. A significant component of the research involves active recruitment and mentoring of students from under-represented areas into the undergraduate and graduate training programs. These studies will add to our basic understanding of the dynamic processes whereby the macromolecular machines of gene expression operate in performing their biological functions. Potential benefits range from an improved understanding of the molecular level interface between chemical and biological behavior, to new general insights about the underlying principles of nano-machines.

该奖项属于生命过程化学 (CLP) 项目，支持俄勒冈大学 Andrew H. Marcus 教授对 DNA 聚合酶和解旋酶的分子机制进行基础研究，这两种蛋白质-DNA 复合物是制定和调节基因表达的生物过程的基本要素。成对的荧光核酸碱基类似物在双链和引物模板 DNA 的分叉和连接处取代天然核苷酸。裸DNA构建体的偏振荧光波动测量将揭示有关碱基类似位点的相对偶极角的信息，以及它们在微秒到亚秒时间尺度上的相对运动。后续实验将在蛋白质-DNA 复合物上进行，以直接探测复制过程关键步骤中蛋白质酶位点的机制。这些研究将提供有关 DNA 合成过渡态途径的详细信息，并研究复制保真度中可能存在的中间态。该研究的一个重要组成部分涉及积极招募和指导来自代表性不足地区的学生进入本科生和研究生培训项目。这些研究将增加我们对基因表达大分子机器执行其生物学功能的动态过程的基本理解。潜在的好处包括提高对化学和生物行为之间分子水平界面的理解，以及对纳米机器基本原理的新的一般见解。