Sensing mechanisms for CO and other small molecules

CO 和其他小分子的传感机制

• 批准号: 7474621
• 负责人: GARY Paul ROBERTS
• 金额: $ 38.71万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7474621
• 关键词: Address; Aerobic; Air; Binding; Binding Sites; Biological Assay; Biological Models; Carbon monoxide dehydrogenase; Class; Cyclic AMP; DNA; DNA Binding Domain; Family; Family member; Gene Expression; Genes; Genetic Engineering; Genomics; Heme; Homologous Gene; In Vitro; Ligands; Metabolism; Methodology; Methods; Numbers; Pathway interactions; Physiological; Physiology; Prokaryotic Cells; Property; Proteins; Rest; Role; Specificity; Structure; System; Variant; Work; base; cysteine rich protein; genome sequencing; insight; interest; member; novel; oxidation; response; sensor; small molecule; success; tool

DESCRIPTION (provided by applicant): Genomic sequencing has shown that the CRP/CooA superfamily of proteins is quite large and must regulate a variety of disparate functions. However, only a modest number of members of this superfamily are understood in terms of their effectors and their physiological role. We have had great success in analyzing the response of CooA to CO binding and the basis of its specificity for CO. We have already extended some of those insights to provide a better understanding of the response of CRP itself to its effector, cAMP. Nevertheless, some key issues remain that we will address in Aim I of this proposal, including (i) the basis for repositioning of the DNA-binding domain in response to effector; (ii) the degree to which the each protein's ligand specificity is indirectly altered by properties physically unlinked to the effector-binding site; and (iii) the identification of the structures for each protein that are necessary for a full understanding of their responses to effector. In another aim of the proposal, we will exploit our understanding of CRP and CooA to develop novel and general methods for understanding the effectors and the physiological roles of other members of the superfamily that have been identified through genome sequencing. In the course of our work on CooA, we have found a completely different CO-sensing transcriptional factor in R. rubrum and other bacterial species, which we have termed RcoM (regulator of CO metabolism). By a variety of criteria RcoM appears to be a CO-sensor that regulates the expression of aerobic CO dehydrogenase systems. The RcoM family contains heme, but is not homologous to CooA. Its effector-binding domain is homologous to that of other heme-containing PAS domains and its DNA-binding domain, termed a LytTR domain, is distributed broadly in prokaryotes, yet its function is poorly understood. We have already developed purification methods, an in vitro functional assay and identified one heme ligand in RcoM. We will exploit these tools and the methodology we developed for CooA to analyze CO responsiveness in this novel family of CO sensors.

描述(由申请人提供)：基因组测序显示，CRP/CooA超家族蛋白相当大，必须调节各种不同的功能。然而，就效应器和生理作用而言，这个超家族中只有少量成员被了解。我们已经成功地分析了CooA对CO结合的响应及其对CO的特异性基础。我们已经扩展了其中的一些见解，以更好地了解CRP本身对其效应物cAMP的反应。然而，一些关键问题仍然是我们将在本提案的目标I中解决的，包括：(I)DNA结合结构域对效应器的反应的重新定位的基础；(Ii)每个蛋白质的配基的特异性被物理上与效应器结合部位无关的性质间接改变的程度；以及(Iii)对每个蛋白质的结构的鉴定，这些结构对于充分了解它们对效应器的反应是必要的。在该提案的另一个目的中，我们将利用我们对CRP和CooA的理解来开发新的和通用的方法，以了解已通过基因组测序确定的超家族其他成员的效应器和生理作用。在对CooA的研究过程中，我们发现了一种在红色杆菌和其他细菌中完全不同的CO感知转录因子，我们称之为RCom(CO代谢调节因子)。从各种标准来看，RCom似乎是一个CO感受器，调节有氧CO脱氢酶系统的表达。RCom家族含有血红素，但与CooA不是同源的。它的效应器结合域与其他含血红素的PAS结构域同源，其DNA结合域被称为LytTR域，广泛分布于原核生物中，但其功能尚不清楚。我们已经开发了纯化方法，体外功能测试，并在RCom中鉴定了一个血红素配体。我们将利用这些工具和我们为CooA开发的方法来分析这一新型CO传感器家族中的CO响应性。