Sensing mechanisms for CO and other small molecules

描述（由申请人提供）：基因组测序表明，CRP/CooA蛋白超家族相当大，必须调节各种不同的功能。然而，这个超家族中只有少数成员在其效应和生理作用方面被理解。我们在分析CooA对CO结合的反应及其对CO的特异性基础方面取得了巨大的成功。我们已经扩展了一些见解，以更好地理解CRP本身对其效应物cAMP的反应。然而，一些关键问题仍然存在，我们将在本提案的目标1中解决，包括(I)响应效应物的dna结合结构域重新定位的基础；（ii）每种蛋白质的配体特异性被物理上与效应结合位点无关的性质间接改变的程度；（iii）鉴定每个蛋白质的结构，这是充分了解它们对效应物的反应所必需的。在该提案的另一个目标中，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来理解通过基因组测序确定的超家族其他成员的效应物和生理作用。在CooA的研究过程中，我们在红毛鼠和其他细菌中发现了一种完全不同的CO感应转录因子，我们将其命名为RcoM （regulator of CO metabolism）。通过各种标准，RcoM似乎是一个CO传感器，调节有氧CO脱氢酶系统的表达。RcoM家族含有血红素，但与CooA不同源。其效应结合域与其他含血红素的PAS结构域同源，其dna结合域LytTR结构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，体外功能测定，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种新型CO传感器家族的CO响应性。

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.