MOLECULAR BASIS FOR CO SENSING AND RESPONSE

暴露于一氧化碳（CO）的红杜鹃（Rhodocellum rubrum）刺激 一组基因的表达，其产物将CO氧化为CO2， 伴随的H+还原成H2。这种反应受COOA的调节 （CO-氧化激活剂）基因产物，CooA。各种基因和 生理结果表明，CooA是一个不寻常的成员， 重要的cAMP受体蛋白（CAP）家族的转录效应子。 特别是，在CAP结构上模拟CooA序列表明， 与cAMP结合同源的区域中的金属簇结合基序 CAP的网站蛋白质中的这种簇通常是CO结合的靶标。 这项研究将阐明CooA介导的机制， R.红色的CO的存在。 第一个具体目标将是纯化CooA，因为这将是 对于详细了解其在CO传感中的作用至关重要， 反应同时，我们将开发一种体外功能测定法， CooA及其过表达的方案。这些工具将用于 与我们在厌氧蛋白纯化方面的专业知识相结合， 功能性CoA的量。 第二个具体目标将直接检查CO-sensing域， CooA.将分析纯化的蛋白质中金属的存在， 金属簇。CooA在CO存在和不存在下的光谱 将被审查。结果将表明的数量和架构 在CO传感基序的金属，这将进一步研究，通过 使用定点诱变和随后的生化分析 一些变异的CooA基因。 最终目标是描述CooA对CO的反应。 涉及检查构象和低聚状态的 CooA，以及DNA结合和转录激活的参数 在存在和不存在CO的情况下。将发现反应改变的突变体 或者通过基于CAP范例的定向诱变，或者通过 已经开发的直接选择方案。 这项工作的结果将是深入了解的分子基础， 生物CO识别以及对CAP/FNR的更好理解 家人CO结合的阐明对于我们理解 一氧化碳作为污染物的影响以及它作为一种 生物信号分子

Exposure of Rhodospirillum rubrum to carbon monoxide (CO) stimulates the expression of a set of genes whose products oxidize CO to CO2 with concomitant reduction of H+ to H2. This response is regulated by the cooA (CO-oxidation Activator) gene product, CooA. A variety of genetic and physiological results indicate that CooA is an unusual member of the important cAMP Receptor Protein (CAP) family of transcriptional effectors. In particular, modeling the CooA sequence on the CAP structure suggests a metal cluster-binding motif in the region homologous to the cAMP binding site of CAP. Such clusters in proteins are often targets for CO-binding. The proposed research will elucidate the mechanism by which CooA mediates the response of R. rubrum to the presence of CO. The first specific aim will be to purify CooA, since this will be essential for any detailed understanding of its role in CO-sensing and response. Simultaneously we will develop an in vitro functional assay for CooA and a protocol for its overexpression. These tools will be used in concert with our expertise in anaerobic protein purification to purify mg quantities of functional CooA. The second specific aim will directly examine the CO-sensing domain of CooA. The purified protein will be analyzed for the presence of metals and metal clusters. The spectra of CooA both in the presence and absence of CO will be examined. The results will indicate the number and architecture of the metals at the CO-sensing motif, which will be further examined through the use of site-directed mutagenesis and subsequent biochemical analysis of a few selected mutant versions of CooA. The final aim will characterize the response of CooA to CO. This will involve examination of the conformation and oligomerization states of CooA, as well as parameters of DNA binding and transcriptional activation in the presence and absence of CO. Response-altered mutants will be found either through directed mutagenesis based on the CAP paradigm or through direct selection schemes that have already been developed. The result of this work will be an insight into the molecular basis for biological CO recognition as well as improved understanding of the CAP/FNR family. The elucidation of CO binding is important to our understanding of the effects of CO as a pollutant as well as its possible role as a biological signal molecule.