The Molecular Basis for CO-Sensing and Response

描述（由申请人提供）：红红螺旋菌通过表达一组基因来响应环境中一氧化碳（CO）的存在，这些基因的产物将CO氧化为CO2。CooA是一种能够感知还原条件和CO的存在并通过激活CO依赖性转录做出反应的蛋白质。CooA是一种同型二聚体，含血红素的蛋白，是CRP/FNR激活剂家族的成员。CooA存在于活性和非活性形式之间的平衡状态，只有CO与CooA的血红素结合才能使这种平衡向活性形式转变。我们的工作假设是CO结合通过重新定向血红素导致这种激活，这反过来又导致相邻的长α螺旋在较暗的界面上重新定位。这种螺旋重定位通过蛋白质传递CO结合信号，稳定dna结合域的活性构象。目前的提案将测试这一工作模式的许多方面。这一分析将涉及各种突变分析，既包括根据过去的结果进行的有针对性的变化，也包括与强大的遗传筛选相结合的随机突变。具有有趣特性的CooA变异将在体外进行纯化和功能和光谱表征，以确定其特性的分子基础。通过这种方法，我们将更好地定义(i) CO活化CooA特异性的分子基础；（ii） CooA内信号转导机制的性质；（iii）活性和非活性状态平衡的生化基础；（iv）激活蛋白质的构象变化背后的驱动力；(v) CooA与RNA聚合酶和特定DNA序列相互作用的基础。在最近的资助期内，我们对CooA的理解急剧增加，使其成为理解CRP家族中共同行为的优秀模型系统，其中一些参与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与血红蛋白、P-450和鸟酰环化酶等更多医学相关的血红蛋白相比，具有明显的技术优势，对CooA的分析也将有助于深入了解血红素配体在其他蛋白质中的功能重要性。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可以作为一种特别容易处理的模型系统来理解生物系统中CO的感知。

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.