The Molecular Basis for CO-Sensing and Response

描述（由申请人提供）：红杜鹃通过表达一组基因来响应其环境中一氧化碳（CO）的存在，所述基因的产物将CO氧化为CO2。CooA是感知还原条件和CO的存在并通过激活CO依赖性转录来响应的蛋白质。CooA是一种含血红素的同型二聚体蛋白，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中，并且只有CO与CooA的血红素的结合将该平衡向活性形式移动。我们的工作假设是，CO结合通过重定向血红素而引起这种激活，这反过来又引起邻近的长α螺旋在较暗界面处的重新定位。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结合结构域的活性构象。本提案将检验这一工作模式的许多方面。该分析将涉及各种突变分析，既包括由过去结果提供信息的靶向变化，也包括随机诱变与强大的遗传筛选。具有有趣特性的CooA变体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。通过这一点，我们将更好地定义（i）由CO激活CooA特异性的分子基础;（ii）CooA内信号转导机制的性质;（iii）活性和非活性状态之间平衡的生物化学基础;（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。在最近的资助期内，我们对CooA的理解急剧增加，使其成为理解CRP家族内共享行为的极好模型系统，其中一些参与调节其他生物体中的毒力。作为一种易于评估的转录调节因子，CooA与血红蛋白、P-450和鸟苷酸环化酶等医学相关血红素蛋白相比具有明显的技术优势，其分析将为了解血红素配体在其他蛋白中的功能重要性提供帮助。最后，由于越来越多的证据CO作为信使分子在哺乳动物系统中，CooA作为一个特别容易处理的模型系统，了解CO传感在生物系统中。

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.