MOLECULAR BASIS FOR CO SENSING AND RESPONSE

CO 传感和响应的分子基础

• 批准号: 2192548
• 负责人: GARY Paul ROBERTS
• 金额: $ 17.8万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2192548
• 关键词: DNA binding protein; DNA footprinting; Escherichia coli; Rhodospirillum; SDS polyacrylamide gel electrophoresis; bacterial genetics; bioassay; biological signal transduction; carbon monoxide; chemical binding; conformation; cysteine; gel mobility shift assay; gene expression; genetic transcription; ligands; molecular biology; molecular cloning; mutant; protein purification; site directed mutagenesis; transcription factor; DNA binding protein; DNA footprinting; Escherichia coli; Rhodospirillum; SDS polyacrylamide gel electrophoresis; bacterial genetics; bioassay; biological signal transduction; carbon monoxide; chemical binding; conformation; cysteine; gel mobility shift assay; gene expression; genetic transcription; ligands; molecular biology; molecular cloning; mutant; protein purification; site directed mutagenesis; transcription factor

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.

将黄褐色rubrum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与 伴随H+将H+降低到H2。此响应受COO的调节 （共氧化激活剂）基因产物，COOA。各种遗传和 生理结果表明COOA是不寻常的成员 重要的cAMP受体蛋白（CAP）转录效应子家族。 特别是，对帽结构上的COOA序列进行建模表明A 与cAMP结合的区域中的金属簇结合图案 上限站点。蛋白质中的这种簇通常是共同结合的靶标。 拟议的研究将阐明COOA介导的机制 R. rubrum对CO的存在的响应。 第一个具体目的是净化COOA，因为这将是 详细了解其在共同信号中的作用至关重要 回复。同时，我们将开发用于体外功能测定法 COOA及其过表达的协议。这些工具将用于 与我们在厌氧蛋白纯化方面的专业知识一起纯化Mg 数量的功能性COOA。 第二个特定目的将直接研究 COOA。将分析纯化的蛋白质的金属和 金属簇。在存在和不存在CO的情况下，COOA的光谱 将被检查。结果将指示 共同感应图案的金属将通过 使用位置定向的诱变和随后的生化分析 在一些选定的突变版本的COOA中。 最终目标将表征COOA对CO的反应。这将 涉及检查构象和寡聚状态的 DNA结合和转录激活的COOA以及参数 在存在和不存在CO的情况下。 通过基于盖范式的定向诱变或通过 已经开发的直接选择方案。 这项工作的结果将是对分子基础的见解 生物CO识别以及对CAP/FNR的了解 家庭。阐明CO绑定对我们对我们的理解很重要 CO作为污染物的影响以及其可能的作用 生物信号分子。