PHYSICAL AND GENETIC STUDIES OF REPRESSOR PROTEINS

阻遏蛋白的物理和遗传学研究

• 批准号: 3271148
• 负责人: KATHLEEN S MATTHEWS
• 金额: $ 16.03万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271148
• 关键词: DNA; DNA binding protein; Escherichia coli; X ray crystallography; analytical method; antibody; bacterial genetics; binding proteins; calorimetry; chemical association; chemical reaction; circular DNA; conformation; fluorescence spectrometry; laboratory rabbit; lac operon; mutant; nuclear magnetic resonance spectroscopy; nucleoproteins; point mutation; protein engineering; protein sequence; protein structure function; thermodynamics; tryptophan

This research project is designed to investigate the mechanisms by which proteins recognize specific sequences of DNA and the means by which small ligands modulate the recognition process. The lac and trp repressors from E. coli represent regulatory proteins in the classes of inducible and repressible systems; both of these proteins and their target DNA sequences can be isolated in the quantities required for physical and chemical studies, and the coding regions are available on vectors for site-specific mutagenesis and other genetic manipulations. The specific objectives are: (1) assessment of the roles of specific amino acids and regions of the primary sequences in the function of these repressor proteins. This examination will include generation of site-specific mutants based on the trp repressor x-ray crystallographic structure and on the known chemical, physical and homology data for lac repressor. Interchange of the helix-turn-helix structure implicated in DNA binding between the two repressors will be undertaken. These specifically designed proteins and mutant proteins available from other sources will be characterized in detail. Antibodies to specific segments of the primary sequence of these proteins will be generated, and the effects of antibody-repressor complex formation on functional activities will be measured. (2) Determination of the effects of DNA structure on binding activities. The effects of supercoiling on the binding parameters for repressor-DNA (both operator and nonspecific binding) will be examined. Cross-linking of proteins to DNA will be attempted to assess specific regions of the protein which are in close proximity to DNA. (3) Thermodynamic studies of protein structure and function. High pressure dissociation of the repressor proteins and effects of ligands on this process will provide information about the strength of inter-subunit contacts and alterations in these contacts by ligand binding. Calorimetric measurements of protein denaturation (DSC) and ligand binding will be undertaken to assess the thermodynamic properties of the proteins and their interactions. (4) Determination of protein structure and conformational changes. FLuorescence spectroscopy of intrinsic and extrinsic fluorophores will be measured, and NMR spectroscopy will be utilized to examine the binding properties of the trp repressor. The correlation of data obtained from these studies on two different systems with data available in the literature will expand our knowledge of genetic regulation, an essential function in all living organisms.

该研究项目旨在研究 蛋白质识别DNA的特定序列和小的序列 配体调节识别过程。 LAC和TRP阻遏物来自 大肠杆菌代表诱导和类别中的调节蛋白 可抑制系统；这两种蛋白质及其靶DNA序列 可以隔离物理和化学所需的数量 研究和编码区域可用于特定于网站的向量 诱变和其他遗传操作。 具体目标是： （1）评估特定氨基酸和区域的作用 这些阻遏蛋白功能的主要序列。 这 考试将包括基于地点特异性突变体的生成 TRP阻遏物X射线晶体结构以及已知的化学物质， LAC阻遏物的物理和同源性数据。 交换 螺旋 - 转向螺旋结构与两者之间的DNA结合有关 将进行阻遏物。 这些专门设计的蛋白质和 其他来源可用的突变蛋白将在以 细节。 这些主要序列特定段的抗体 将生成蛋白质，并产生抗体压迫剂复合物的作用 将测量有关功能活动的形成。 （2）确定 DNA结构对结合活性的影响。 效果 在抑制剂-DNA的绑定参数上超过固定（运算符和 将检查非特异性结合）。 蛋白质与DNA的交联 将尝试评估蛋白质的特定区域 接近DNA。 （3）蛋白质结构和 功能。 阻遏蛋白的高压解离和作用 在此过程中配体的配体将提供有关强度的信息 配体中这些触点间的亚基触点和变化 结合。 蛋白质变性（DSC）和 将进行配体结合，以评估 蛋白质及其相互作用。 （4）测定蛋白质 结构和构象变化。 荧光光谱 将测量固有和外在的荧光团，并测量NMR光谱 将用于检查TRP阻遏物的结合特性。 从这些研究获得的两个不同的数据的相关性 文献中可用数据的系统将扩大我们对 遗传调节，在所有生物体中的重要功能。