MOLECULAR BASIS FOR CO SENSING AND RESPONSE

红色红螺菌暴露于一氧化碳(CO)刺激 一组基因的表达，其产物将CO氧化为CO2 伴随地将H+还原为H2。这种反应是由CooA调节的 (共氧化激活剂)基因产物CooA。一系列的基因和 生理结果表明，CooA是一种不寻常的成员 重要的cAMP受体蛋白(CAP)家族转录效应。 特别是，在CAP结构上对CooA序列进行建模表明 CAMP结合同源区域中的金属簇结合基序 CAP的站点。蛋白质中的这种簇通常是CO结合的目标。 这项拟议的研究将阐明CooA介导的机制 红色红曲霉对CO存在的反应。 第一个具体目标将是净化CooA，因为这将是 对于详细了解其在一氧化碳传感中的作用和 回应。同时，我们将开发一种体外功能检测方法，用于 CooA及其过度表达的协议。这些工具将用于 与我们在厌氧蛋白纯化方面的专业知识合作提纯镁 功能性CooA的数量。 第二个具体目标将直接检查CO感应域 库阿。纯化的蛋白质将被分析是否存在金属和 金属簇。CO存在和不存在时CooA的光谱 将会被检查。结果将表明， CO感应基元上的金属，将通过 定点突变和后续生化分析的应用 一些精选的CooA突变版本。 最终目标将表征CooA对CO的响应。这将是 包括对其构象和齐聚状态的研究 CooA，以及DNA结合和转录激活的参数 在CO存在和不存在的情况下。将会发现反应改变的突变体 通过基于CAP范例的定向突变或通过 已经制定的直接选拔计划。 这项工作的结果将是洞察分子基础 生物CO识别以及对CAP/FNR更好的理解 一家人。CO结合的阐明对于我们理解 CO作为一种污染物的影响及其可能的作用 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.