Quantitative Studies of the Biotin Regulatory System

生物素调节系统的定量研究

• 批准号: 7417426
• 负责人: DOROTHY BECKETT
• 金额: $ 25.34万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2009-08-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7417426
• 关键词: 5' -AMP-biotin; Acetyl-CoA Carboxylase; Anabolism; Binding; Binding Proteins; Biological Models; Biotin; Cell Communication; Cell Surface Receptors; Cell physiology; Cells; Class; Complex; Condition; Coupled; Coupling; Crystallography; DNA Binding; DNA-Binding Proteins; Defect; Development; Digestion; Enzymes; Escherichia coli; Gene Expression; Genetic; Genetic Transcription; Growth; Heterodimerization; Homodimerization; Investigation; Kinetics; Lactose; Ligands; Link; Mass Spectrum Analysis; Measurement; Measures; Metabolic; Metabolism; Methods; Molecular; Molecular Mimicry; N-terminal; Nutrient; Operon; Organism; Pathway interactions; Physiological; Post-Translational Protein Processing; Process; Production; Prokaryotic Cells; Property; Proteins; Rate; Reaction; Regulation; Research Personnel; Role; Signal Transduction; Site; Solutions; Structure; Surface; Surface Plasmon Resonance; System; Thermodynamics; Transcription Initiation; Transcription Regulatory Protein; Tryptophan; analog; base; biotin repressor; design; extracellular; insight; monomer; mutant; programs; protein protein interaction; response; self assembly; stopped-flow fluorescence; transcription factor

Transcription of genetic information in all organisms must be responsive to extracellular and intracellular signals such as levels of metabolytes, cell-cell interactions, and occupancy of cell surface receptors by ligands. Multifunctional transcription regulatory proteins provide an efficient means of directly linking these signals to the transcription initiation process. Quantitative studies of these transcription factors will reveal the molecular switching mechanisms that enable intimate coupling of an array of physiological signals to genetic regulation. In E.coli, production of the nutrient, biotin, is linked to cellular demand by BirA, a multi-functional transcription factor. BirA is an allosteric DMA binding protein and the enzyme that catalyzes posttranslational addition of biotin to a carboxylase. The intermediate in the biotin transfer reaction, bio-5'-AMP, also activates the protein for site-specific DNA binding by dramatically enhancing the energetics of BirA homodimerization. Thus, elucidation of the consequences of bio-5'-AMP binding for the structural and dynamic properties of the represser monomer will provide insight into the pathway of allosteric activation. These studies will employ stopped-flow fluorescence measurements of effector binding and structural analysis of the complexes using H/D exchange coupled to mass spectrometry. The functional switch of BirA from transcription represser to enzyme is hypothesized to occur via switching of protein partners. Moreover, as the same surface of the represser is proposed to be utilized for the two protein:protein interactions, molecular mimicry is anticipated to contribute to the switching process. Thermodynamic and kinetic measurements of the alternative protein:protein interactions will reveal the molecular basis of the use of a single surface for the alternative interactions that are central to this transcriptional switch. Surface Plasmon Resonance, steady-state and stopped-flow fluorescence and quench-flow methods will be used for these measurements. Structural analysis of the relevant complexes will be carried out using x-ray crystallography.

所有生物体中遗传信息的转录必须对细胞外和细胞内信号做出响应，例如代谢物水平、细胞间相互作用以及配体对细胞表面受体的占据。多功能转录调节蛋白提供了一种将这些信号直接连接到转录起始过程的有效方法。对这些转录因子的定量研究将揭示分子转换机制，使一系列生理信号与遗传调控紧密耦合。在大肠杆菌中，营养物质生物素的产生与 BirA（一种多功能转录因子）的细胞需求相关。 BirA 是一种变构 DMA 结合蛋白和催化翻译后 将生物素添加到羧化酶中。生物素转移反应的中间体bio-5'-AMP， 还通过显着增强 BirA 同二聚化的能量来激活蛋白质进行位点特异性 DNA 结合。因此，阐明 bio-5'-AMP 结合对阻抑单体的结构和动态特性的影响将有助于深入了解变构激活途径。 这些研究将采用停流荧光测量效应子结合，并使用 H/D 交换与质谱联用对复合物进行结构分析。假设 BirA 从转录抑制蛋白到酶的功能转换是通过蛋白质伴侣的转换而发生的。此外，由于阻遏物的相同表面被提议用于两种蛋白质：蛋白质相互作用，因此分子拟态预计将有助于转换过程。替代蛋白质：蛋白质相互作用的热力学和动力学测量将揭示使用单一表面进行替代相互作用的分子基础，而替代相互作用是这种转录开关的核心。表面等离子共振、稳态和停流荧光以及猝灭流方法将用于这些测量。将使用 X 射线晶体学对相关配合物进行结构分析。