Sensing mechanisms for CO and other small molecules

CO 和其他小分子的传感机制

• 批准号: 7315183
• 负责人: GARY Paul ROBERTS
• 金额: $ 40.91万
• 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/7315183
• 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）：基因组测序表明，蛋白质的CRP/COOA超家族非常大，必须调节各种不同的功能。但是，从其效应子及其生理作用方面，只了解了这个超家族的数量适中的成员。我们在分析COOA对COBIN的响应及其对CO的特异性的基础方面取得了巨大的成功。我们已经扩展了其中一些见解，以更好地了解CRP本身对其效应子Camp的响应。尽管如此，我们将在该提案的目标中解决一些关键问题，包括（i）重新定位DNA结合域以响应效应子的基础； （ii）每种蛋白质的配体特异性的程度通过物理上未与效应子结合位点进行物理不链接的特性间接改变； （iii）鉴定每种蛋白质的结构，这是对它们对效应子的反应的充分理解所必需的。为了提案的另一个目的，我们将利用对CRP和COOA的理解来开发新颖的和一般方法，以理解通过基因组测序确定的超级家族其他成员的效应子和生理作用。在我们在COOA上的工作中，我们发现了R. rubrum和其他细菌物种的完全不同的共感敏感性转录因子，我们称它们为RCOM（CO代谢的调节剂）。根据多种标准，RCOM似乎是调节有氧CO脱氢酶系统表达的共同传感器。 RCOM家族包含血红素，但与COOA并非同源。它的效应结合结构域与其他含血红素的PAS结构域及其DNA结合结构域的效应域是同源的，称为LYTTR结构域，该结构域分布在原核生物中，但其功能知之甚少。我们已经开发了纯化方法，一种体外功能分析，并在RCOM中鉴定了一种血红素配体。我们将利用这些工具和为COOA开发的方法来分析这个新型CO传感器家族中的CO响应能力。