Molecular Origins of Specificity in Protein-Nucleic Acid Interactions

蛋白质-核酸相互作用特异性的分子起源

• 批准号: 0136094
• 负责人: Jannette Carey
• 金额: $ 37.5万
• 依托单位: Princeton University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0136094&HistoricalAwards=false
• 关键词: Molecular; Origins; Specificity; Protein; Nucleic

The molecular mechanisms by which small-molecule allosteric effectors activate gene-regulatory proteins for specific DNA binding are largely unknown. Understanding these mechanisms should provide general insights into molecular recognition, and inform our understanding of the biology of the molecules involved in these processes. The overall goal of the present work is to achieve an understanding of allosteric activation that accounts for both structural and energetic features of ligand binding and explains DNA specificity in the arginine repressor system of E. coli, ArgR. A combined biochemical and biophysical approach will be used to establish the quantitative foundations for a comprehensive understanding of the molecular and biological significance of ArgR-ligand interactions. The cornerstone of this approach is careful measurement of ligand-binding equilibria including determination of affinity, stoichiometry, and cooperativity. The goal in making these measurements is to define the ligand-occupancy states of the protein as a function of ligand concentration. This information is then used to set the conditions for studies of protein function and structure over the range of relevant ligand-occupancy states. This project has four aims. 1. Quantitative analysis of L-arg binding to ArgR using isothermal titration, analytical ultracentrifugation, and NMR. 2. Analysis of the effects of ligand-occupancy state on ArgR function in transcription and recombination. 3. Evaluation of changes in structure and dynamics as a function of ligand-occupancy state, using proteolytic cleavage and NMR. 4. Examination of ATP binding to evaluate whether this ligand confers functional or structural consequences for ArgR. These biochemical and biophysical experiments should provide the quantitative foundations for understanding the molecular and biological mechanisms of ArgR function, and should help to bring the functional and structural pictures of ArgR allosteric activation into a common focus. Biomolecules have difficult jobs to do. Many of them must recognize one ligand from an intracellular sea of similar ligands, and respond in a physiologically appropriate way. The range of molecular strategies used to execute complex cellular functions is probably very broad, but few examples have been fully characterized. An in-depth understanding of molecular strategies is fundamental to many areas of chemistry and biology. This research is aimed at expanding our general understanding of the mechanisms used by proteins to recognize and respond to small ligands that can potentiate their function.

小分子变构效应物激活特定DNA结合的基因调控蛋白的分子机制在很大程度上是未知的。了解这些机制将提供对分子识别的一般见解，并告知我们对这些过程中涉及的分子生物学的理解。目前工作的总体目标是实现对变构激活的理解，该激活解释了配体结合的结构和能量特征，并解释了大肠杆菌精氨酸抑制系统（ArgR）中的DNA特异性。将采用生物化学和生物物理相结合的方法，为全面了解argr -配体相互作用的分子和生物学意义建立定量基础。这种方法的基础是仔细测量配体结合平衡，包括亲和力、化学计量和协同性的测定。进行这些测量的目的是确定蛋白质的配体占用状态作为配体浓度的函数。然后，这些信息被用于在相关配体占用状态范围内为蛋白质功能和结构的研究设定条件。这个项目有四个目标。1. L-arg与ArgR结合的定量分析，使用等温滴定，分析超离心和核磁共振。2. 配体占用状态对ArgR转录和重组功能的影响分析。3. 利用蛋白水解裂解和核磁共振评价配体占有状态下结构和动力学的变化。4. 检查ATP结合以评估该配体是否对ArgR具有功能或结构上的影响。这些生物化学和生物物理实验将为了解ArgR功能的分子和生物学机制提供定量基础，并有助于将ArgR变构激活的功能和结构图片纳入共同关注的焦点。生物分子有困难的工作要做。它们中的许多必须从细胞内相似的配体海洋中识别出一种配体，并以生理上适当的方式作出反应。用于执行复杂细胞功能的分子策略的范围可能非常广泛，但很少有例子得到充分表征。深入了解分子策略是化学和生物学许多领域的基础。这项研究旨在扩大我们对蛋白质识别和响应可以增强其功能的小配体的机制的一般理解。