Probing Reaction Pathways in Protein-DNA Complexes by Two-Dimensional Fluorescence Spectroscopy and Single-Molecule Correlation Spectroscopy

通过二维荧光光谱和单分子相关光谱探测蛋白质-DNA 复合物的反应途径

• 批准号: 1307272
• 负责人: Andrew Marcus
• 金额: $ 49万
• 依托单位: University of Oregon Eugene
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1307272&HistoricalAwards=false
• 关键词: Probing; Reaction; Pathways; Protein; DNA

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 to investigate the local base conformations and conformational dynamics at pre-selected sites within functional DNA-protein replication complexes and to determine key structural end-states, intermediates, and transition states along enzymatic reaction pathways of these complexes. These experiments employ DNA constructs containing two adjacently positioned fluorescent nucleic acid base analogue probes, which absorb in a spectral region where DNA and proteins are transparent. Coupling between the electronic states of the dimer probe residues produces signature spectral features, which are used to infer local nucleic acid base conformation and time-dependent local motions, which is information central to understanding the mechanisms by which these complexes carry out their specialized functions. These studies will be carried out using two-dimensional fluorescence spectroscopy (2D FS, a fluorescence-detected version of 2D electronic coherence spectroscopy) to elucidate analogue dinucleotide probe conformation, and single-molecule Forster resonance energy transfer (smFRET) and fluorescence-detected linear dichroism (smFLD) to monitor conformational dynamics on time scales ranging between tens-of-microseconds to tens-of-seconds. The work supported will add to our basic understanding of the dynamic processes whereby the macromolecular machines of gene regulation and expression perform 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. A significant component of the research and educational activities also involves mentoring the next generation of scientists, and encouraging the participation of students from under-represented groups. These activities include i) the involvement of undergraduate students in research projects, and providing them with guidance to develop professional careers; ii) the development of new courses at the undergraduate and graduate levels within the UO Chemistry Department; and iii) outreach to local high school students through the creation of advanced placement chemistry courses that are taught on furlough days. Graduate and postdoctoral students working on these projects will benefit from cross-disciplinary training in physical chemistry, biological sciences, and theory, enhancing their future career opportunities.

生命过程化学(CLP)项目的这一奖项支持俄勒冈大学的安德鲁·H·马库斯教授开展基础研究，以研究功能性DNA-蛋白质复制复合体中预选位置的局部碱基构象和构象动力学，并确定这些复合体沿酶反应路径的关键结构终态、中间体和过渡态。这些实验使用的DNA构建包含两个相邻定位的荧光核酸碱基模拟探针，它们在DNA和蛋白质透明的光谱区域吸收。二聚体探针残基的电子态之间的耦合产生了特征光谱特征，这些特征被用来推断局部核酸碱基构象和依赖时间的局部运动，这是理解这些复合体执行其专门功能的机制的核心信息。这些研究将使用二维荧光光谱(2D FS，2D电子相干光谱的荧光检测版本)来阐明模拟二核苷酸探针的构象，并使用单分子Forster共振能量转移(SmFRET)和荧光检测线性二色性(SmFLD)来监测从几十微秒到几十秒的时间尺度上的构象动力学。所支持的工作将增加我们对基因调控和表达的大分子机器执行其生物功能的动态过程的基本理解。潜在的好处包括更好地理解化学和生物行为之间的分子水平界面，以及对纳米机器基本原理的新的一般性见解。研究和教育活动的一个重要组成部分还涉及指导下一代科学家，并鼓励来自代表性不足群体的学生参与。这些活动包括i)让本科生参与研究项目，并为他们提供职业发展的指导；ii)在UO化学系内开发本科生和研究生的新课程；以及iii)通过在休息日教授的高级选修化学课程向当地高中生推广。从事这些项目的研究生和博士后将受益于物理化学、生物科学和理论方面的跨学科培训，从而增加他们未来的职业机会。