Structural and Functional Characterization of Cyanoglobin

Lecomte，Juliette0091182蓝细菌是一种含氧原核生物，现在已知能产生一种与某些细菌和原生动物血红蛋白相关的血红蛋白(Hb)。虽然在固氮蓝藻中，血红蛋白似乎保护固氮酶复合体免受氧化损伤，但在非固氮蓝藻中，如聚球藻。PCC6803中，血红蛋白的作用仍然是个谜。本项目的近期目标是研究聚球藻HB的结构与活性之间的关系。生物物理方法(核磁共振光谱、光谱、离心法、氧化还原电位测量)和生化方法(遗传操作、免疫染色技术、表型鉴定、定点突变)将被应用于1)体内研究聚球藻HB；2)确定其在溶液中的三维结构；3)分析其血红素配位和电化学性质的分子决定因素。将与其他血红蛋白进行比较，以发展对蓝藻球蛋白中血红素活性的原子水平的了解，并将这些蛋白质置于其进化的上下文中。珠蛋白家族的蛋白质在几乎所有生物界都具有代表性。在脊椎动物中，血红蛋白和肌红蛋白负责氧气的运输和储存，但在原始生物体中，珠蛋白的功能包括清除氧气、输送、传感和解毒以及管理亚硝化应激。像集胞藻这样的蓝藻可以像其他原核生物一样进行实验操作，并受到遗传方法的影响。因此，它们非常适合于古代血红蛋白的研究。对蓝藻血红蛋白功能的描述有望提供对全球碳和氮循环中必不可少的生物生物化学的基本见解。该项目将为生物化学课程和核磁共振课程等教学活动提供支持。它将为本科生和研究生提供良好的生物物理和生化方法培训机会，并促进多学科互动。

Lecomte, Juliette0091182Cyanobacteria are oxygenic prokaryotes now known to produce a hemoglobin (Hb) related to certain bacterial and protozoan hemoglobins. Although in nitrogen-fixing cyanobacteria, hemoglobin appears to protect the nitrogenase complex from oxidative damage, in non-nitrogen-fixing cyanobacteria, such as Synechocystis sp. PCC 6803, the role of hemoglobin remains a mystery. The immediate goal of this project is to study the relationship between structure and reactivity in the hemoglobin of Synechocystis.Biophysical methods (NMR spectroscopy, optical spectroscopy, centrifugation, redox potential measurements) and biochemical methods (genetic manipulations, immunostaining techniques, phenotypic characterization, site-directed mutagenesis) will be applied to 1) investigate Synechocystis Hb in vivo; 2) determine its three-dimensional structure in solution; and 3) analyze the molecular determinants of its heme coordination and electrochemical properties. Comparison will be made to other hemoglobins to develop an atomic level understanding of heme reactivity in cyanobacterial globins and situate these proteins in their evolutionary context.The globin family of proteins has representatives in practically all kingdoms of organisms. In vertebrates, hemoglobins and myoglobins are responsible for the transport and storage of oxygen but in primitive organisms, the functional roles of globins include oxygen scavenging, delivery, sensing, and detoxification, and management of nitrosative stress. Cyanobacteria such as Synechocystis can be manipulated experimentally like other prokaryotes and subjected to genetic methods. Thus they are ideally suited for studies of ancient hemoglobins. A description of the function of cyanobacterial hemoglobins is expected to provide fundamental insight in the biochemistry of organisms essential in the global cycling of carbon and nitrogen.The project will serve as a support in teaching activities such as a biological chemistry course and an NMR course. It will provide excellent opportunities for undergraduate and graduate student training in biophysical and biochemical methods and foster multidisciplinary interactions.