Molecular Origins of Specificity in Protein-Nucleic Acid Interactions

小分子变构效应物激活基因调节蛋白以特异性结合DNA的分子机制在很大程度上是未知的。了解这些机制应该提供一般的见解分子识别，并告知我们的生物学的分子参与这些过程的理解。本工作的总体目标是实现对变构激活的理解，该变构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阻遏系统中的DNA特异性。coli、ArgR.一个生物化学和生物物理相结合的方法将被用来建立一个全面的了解ArgR-配体相互作用的分子和生物学意义的定量基础。这种方法的基石是仔细测量配体结合平衡，包括确定亲和力，化学计量和协同性。进行这些测量的目标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条件的蛋白质的功能和结构的研究范围内的相关配体占用状态。该项目有四个目标。 1.使用等温滴定、分析超离心和NMR定量分析L-arg与ArgR的结合。 2.分析配体占据状态对ArgR转录和重组功能的影响。 3.使用蛋白水解裂解和NMR评估作为配体占据状态的函数的结构和动力学变化。 4.检查ATP结合，以评估该配体是否赋予ArgR功能或结构后果。 这些生物化学和生物物理实验应该提供定量的基础，了解ArgR功能的分子和生物学机制，并应有助于把ArgR变构激活的功能和结构图片到一个共同的焦点。 生物分子有困难的工作要做。 它们中的许多必须从类似配体的细胞内海洋中识别一种配体，并以生理上适当的方式应答。 用于执行复杂细胞功能的分子策略的范围可能非常广泛，但很少有例子被充分表征。 对分子策略的深入理解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。

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.