Structural and Functional Characterization of Cyanoglobin

血红蛋白超家族在所有生物中都有代表。在脊椎动物中，血红蛋白及其近亲肌红蛋白负责分子氧的运输和储存。这两种高度特化的蛋白质的三维结构和功能之间的关系已经被广泛探索并得到了很好的理解。在无脊椎动物中，血红蛋白表现出更广泛的生理作用，反映了血红蛋白折叠进化、氧气利用和血红素反应性的基本方面。然而，对于这些蛋白质，结构和功能之间的联系是不完全确定的，目前还不可能根据氨基酸序列预测化学性质。这种知识的缺乏限制了基因组测序数据的有用性和对进化关系的解释。该项目的目的是研究非固氮蓝藻聚囊藻sp. PCC 6803和聚囊球菌sp. PCC 7002的原始血红蛋白。它们都是六配位血红蛋白，利用两种组氨酸来连接铁离子，并且都经历了一个简单的翻译后血红素基团的附着，这显然是它们独有的。生物物理方法（体外和体内核磁共振光谱，光谱学）和生化方法（定点诱变，遗传操作，表型表征）将应用于研究聚珠球菌血红蛋白在体内的作用；测定聚囊菌和聚囊球菌蛋白经过血红素修饰前后在溶液中的三维结构；并分析了血红素配位和反应性的分子决定因素。将与其他无脊椎动物血红蛋白进行比较，以发展对化学和生化特性的原子水平的理解。该项目的研究部分为研究生和本科生提供培训机会，并通过以蓝藻生理学和生物化学为重点的合作，建立了化学系与生物化学和分子生物系之间多学科互动的机制。该项目还包括三个具体的教育组成部分，将进一步加强其更广泛的影响。首先是为高等本科生物化学课程开发三维可视化模块，并在本课程中包含与血红蛋白研究相关的主题。这样做的目的是使材料变得生动，并鼓励化学专业的学生追求与生命科学相关的职业。二是针对研究生开设沟通技巧的短期课程。重点将放在如何向普通观众传达复杂的科学概念和问题。三是继续组织并增加结构生物学跨部门研讨会的频率，演讲者为高级学生。这些活动旨在为本科生、研究生和博士后在学术界、工业界和政府的职业生涯做好准备。

The hemoglobin superfamily of proteins has representatives in all kingdoms of organisms. In vertebrates, hemoglobin and its close relative myoglobin are responsible for the transport and storage of molecular oxygen. The relationship between three-dimensional structure and function in these two highly specialized proteins has been explored extensively and is well understood. In invertebrates, hemoglobins exhibit a broader range of physiological roles reflecting essential aspects of globin fold evolution, oxygen utilization, and heme reactivity. For these proteins, however, the connection between structure and function is incompletely defined and it is not yet possible to anticipate chemical properties on the basis of the amino acid sequences. This lack of knowledge limits the usefulness of genome sequencing data and the interpretation of evolutionary relationships. The goal of the project is to investigate primitive hemoglobins from the non-nitrogen fixing cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Both are hexacoordinate hemoglobins utilizing two histidines to ligate the iron ion, and both undergo a facile post-translational attachment of the heme group apparently unique to them. Biophysical methods (NMR spectroscopy in vitro and in vivo, optical spectroscopy) and biochemical methods (site-directed mutagenesis, genetic manipulations, phenotypic characterization) will be applied to investigate the role of Synechococcus hemoglobin in vivo; determine the three-dimensional structure of Synechocystis and Synechococcus proteins in solution before and after they have undergone the heme modification; and analyze the molecular determinants of heme coordination and reactivity. Comparison will be made to other invertebrate hemoglobins to develop an atomic level understanding of chemical and biochemical properties.The research component of the project provides training opportunities for graduate and undergraduate students and, through a collaboration focused on cyanobacterial physiology and biochemistry, establishes a mechanism for multidisciplinary interactions between the Chemistry department and the Biochemistry and Molecular Biology department. The project also contains three specific educational components that will enhance further its broader impact. The first is the development of three-dimensional visualization modules for an advanced undergraduate biological chemistry course and the inclusion in this course of research-related hemoglobin topics. This is designed to bring the material to life and encourage chemistry students to pursue careers with a life sciences slant. The second is the development of a short course in communication skills aimed at graduate students. The emphasis will be placed on how to convey sophisticated scientific concepts and issues to a general audience. The third is to continue the organization and increase the frequency of inter-departmental structural biology seminars, for which the speakers are advanced students. These activities are expected to prepare undergraduate, graduate, and post-doctoral students for professional careers in academia, industry, and government.