PHYSICAL AND GENETIC STUDIES OF REPRESSOR PROTEINS

该研究项目旨在研究其机制 蛋白质识别 DNA 的特定序列以及小分子的识别方式 配体调节识别过程。 lac 和 trp 阻遏物来自 大肠杆菌代表诱导型和可诱导型调节蛋白 可抑制系统；这两种蛋白质及其目标 DNA 序列 可以按照物理和化学所需的量进行分离 研究，编码区可在载体上用于位点特异性 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一级序列。 这个 检查将包括基于位点特异性突变体的生成 色氨酸阻遏物 X 射线晶体结构和已知化学物质， lac 阻遏物的物理和同源性数据。 的互换 螺旋-转角-螺旋结构涉及两者之间的 DNA 结合 将采取抑制措施。 这些专门设计的蛋白质和 从其他来源获得的突变蛋白将被表征为 细节。 针对这些初级序列特定片段的抗体 将产生蛋白质，并且抗体阻遏物复合物的作用 将测量功能活动的形成。 (2) 确定 DNA结构对结合活性的影响。 的影响 阻遏物-DNA 的结合参数上的超螺旋（操作子和 非特异性结合）将被检查。 蛋白质与 DNA 的交联 将尝试评估蛋白质的特定区域 与DNA非常接近。 (3)蛋白质结构和热力学研究 功能。 阻遏蛋白的高压解离及其影响 在此过程中配体的数量将提供有关强度的信息 亚基间接触以及配体对这些接触的改变 绑定。 蛋白质变性 (DSC) 的量热测量和 将进行配体结合以评估热力学性质 蛋白质及其相互作用。 (4)蛋白质的测定 结构和构象的变化。 荧光光谱 将测量内在和外在荧光团，以及核磁共振波谱 将用于检查色氨酸阻遏物的结合特性。 从这些研究中获得的数据的相关性对两个不同的 具有文献中可用数据的系统将扩展我们的知识 遗传调控是所有生物体的重要功能。

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.