Quantitative Studies of the Biotin Regulatory System

所有生物体中遗传信息的转录都必须对细胞外和细胞内的信号做出反应，如代谢物水平、细胞与细胞的相互作用以及配体对细胞表面受体的占用。多功能转录调控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手段。对这些转录因子的定量研究将揭示使一系列生理信号与遗传调控密切耦合的分子开关机制。在大肠杆菌中，营养物质生物素的产生与细胞的需求有关，Bira是一种多功能转录因子。BIRA是一种变构的DMA结合蛋白，是催化翻译后反应的酶 将生物素添加到羧基酶中。生物素转移反应的中间体BIO-5‘-AMP， 还通过显著增强Bira同源二聚的能量学，激活蛋白质与特定部位的DNA结合。因此，阐明BIO-5‘-AMP结合对阻滞剂单体结构和动力学性质的影响将有助于深入了解变构激活的途径。 这些研究将使用停流荧光测量效应器结合，并使用H/D交换与质谱联用对络合物进行结构分析。BIRA从转录抑制子到酶的功能转换被认为是通过蛋白质伙伴的转换而发生的。此外，由于抑制物的同一表面被认为是用于两种蛋白质的相互作用，分子模仿预计有助于切换过程。替代蛋白质的热力学和动力学测量：蛋白质的相互作用将揭示使用单一表面进行替代相互作用的分子基础，这些相互作用是这种转录开关的核心。这些测量将使用表面等离子体激元共振法、稳态和停流荧光法以及猝灭流法。将使用X射线结晶学对相关络合物进行结构分析。

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.