The Molecular Basis for CO-Sensing and Response

描述(申请人提供)：红色红螺菌通过表达一组基因来响应其环境中一氧化碳(CO)的存在，其产物将一氧化碳氧化为二氧化碳。CooA是一种蛋白质，它能感知还原条件和CO的存在，并通过激活CO依赖的转录来做出反应。CooA是一种含有血红素的同源二聚体蛋白，是CRP/FNR激活剂家族的成员。CooA以活性形式和非活性形式之间的平衡存在，只有CO与CooA的血红素结合才使这种平衡向活性形式移动。我们的工作假设是，CO结合通过改变血红素的方向导致这种激活，这反过来又导致相邻的长阿尔法螺旋在较暗的界面上重新定位。这种螺旋重新定位通过蛋白质传递CO结合的信号，以稳定DNA结合区的活性构象。目前的提案将考验这一工作模式的许多方面。这项分析将涉及各种突变分析，既有过去结果提供的有针对性的变化，也有随机突变和强大的基因筛选。具有有趣特性的CooA变体将在体外纯化并进行功能和光谱表征，以确定其特性的分子基础。通过这一点，我们将更好地定义(I)CO激活CooA的特异性的分子基础；(Ii)CooA内信号转导机制的性质；(Iii)活跃状态和非活跃状态之间平衡的生化基础；(Iv)激活蛋白质的构象变化背后的驱动力；以及(V)CooA与RNA聚合酶和特定DNA序列相互作用的基础。在最近的授权期内，我们对CooA的了解急剧增加，使其成为理解CRP家族中共同行为的极佳模型系统，其中一些参与了其他生物体的毒力调节。作为一种易于评估的转录调节因子，CooA与更具医学意义的血红蛋白、P-450和鸟苷酸环化酶相比具有明显的技术优势，其分析也将为深入了解血红素配基在其他蛋白质中的功能重要性提供依据。最后，鉴于越来越多的证据表明CO在哺乳动物系统中是一种信使分子，CooA是一个特别容易处理的模型系统，可以用来理解生物系统中的CO感知。

DESCRIPTION (provided by applicant): Rhodospirillum rubrum responds to the presence of carbon monoxide (CO) in its environment by expressing a set of genes whose products oxidize CO to CO2. CooA is the protein that senses the presence of both reducing conditions and CO and responds by activating CO-dependent transcription. CooA is a homodimeric, heme-containing protein that is a member of the CRP/FNR family of activators. CooA exists in an equilibrium between active and inactive forms, and only the binding of CO to the heme of CooA shifts that equilibrium toward the active form. It is our working hypothesis that CO binding causes this activation by reorienting the heme, which in turn causes a repositioning of the adjacent long alpha helices at the dimmer interface. This helix repositioning transmits the signal of CO binding through the protein to stabilize the active conformation of the DNA-binding domains. The present proposal will test many aspects of this working model. This analysis will involve a variety of mutational analyses, both targeted changes informed by past results, as well as randomized mutageneses coupled with powerful genetic screens. CooA variants with interesting properties will purified and characterized functionally and spectrally in vitro to determine the molecular basis for their properties. Through this we will better define (i) the molecular basis for the specificity of CooA activation by CO; (ii) the nature of the signal transduction mechanism within CooA; (iii) the biochemical basis for the equilibrium between the active and inactive states; (iv) the driving force behind the conformational change that activates the protein; and (v) the basis for the interactions of CooA with RNA polymerase and with specific DNA sequences.The dramatic increase in our understanding of CooA in the recent grant period has made it an excellent model system for understanding the shared behavior within the CRP family, some of which are involved in regulation of virulence in other organisms. As a readily assessed transcriptional regulator, CooA presents distinct technical advantages compared with more medically relevant hemeproteins such as hemoglobin, P-450 and guanylyl cyclase, and its analysis will provide insights into the functional importance of hemeligands in other proteins as well. Finally, given the increasing evidence for CO as a messenger molecule in mammalian systems, CooA serves as a particularly tractable model system for understanding CO sensing in a biological system.