Molecular Origins of Specificity in Protein-Nucleic Acid Interactions

小分子变构效应器激活基因调控蛋白以与特定DNA结合的分子机制在很大程度上尚不清楚。了解这些机制应该提供对分子识别的一般见解，并帮助我们理解参与这些过程的分子的生物学。本工作的总体目标是了解变构激活，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ArgR精氨酸抑制系统中的DNA特异性。将使用生化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奠定定量基础。这种方法的基础是仔细测量配体结合平衡，包括亲和力、化学计量和协作性的确定。进行这些测量的目的是将蛋白质的配体占据状态定义为配体浓度的函数。然后，这些信息被用来设置在相关配体占据状态范围内研究蛋白质功能和结构的条件。这个项目有四个目标。1.等温滴定、分析超速离心法和核磁共振定量分析L-精氨酸与精氨酸受体的结合。2.分析配基占据状态对ArgR转录和重组功能的影响。3.利用蛋白质水解性裂解和核磁共振技术评价配体占据状态对结构和动力学的影响。4.检测ATP结合，以评估该配体是否对ArgR具有功能或结构上的影响。这些生化和生物物理实验将为理解ArgR功能的分子和生物学机制提供定量的基础，并有助于将ArgR变构激活的功能和结构图像集中在一起。生物分子有很难做的工作。它们中的许多人必须从相似配体的细胞内海洋中识别出一个配体，并以生理上合适的方式做出反应。用于执行复杂细胞功能的分子策略的范围可能非常广泛，但很少有例子被完全描述。深入了解分子策略是许多化学和生物学领域的基础。这项研究旨在扩大我们对蛋白质识别和响应小配体的机制的总体理解，这些小配体可以增强它们的功能。

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.