Structural and Dynamic Characterization of 2/2 Hemoglobins

该奖项是根据2009年的美国复苏和再投资法案（公法111-5）资助的。血红蛋白是最有名的氧转运蛋白，负责血液的红色。 然而，血红蛋白蛋白超家族的成员存在于生命的所有三个领域，包括细菌。 对细菌血红蛋白知之甚少，预计许多血红蛋白参与可逆氧结合以外的过程。 该项目将探索细菌血红蛋白的物理化学性质，该血红蛋白属于最近发现的2/2或“截短”血红蛋白超家族谱系。 对于血红素蛋白的反应性特别重要的是涉及辅因子的相互作用和允许小配体（例如氧）到达中心铁的分子运动。 因此，研究的目标是建立连接结构，动力学和反应性的强大的关系。 为此，将分子生物学方法和光谱方法（光学和核磁共振，NMR）的组合应用于一组代表两组遗传学上不同的截短血红蛋白组（组I和组III）和组I内两个不同类别的蛋白质。 (1)在第一个具体的目标，血红蛋白的动力学特性从蓝藻聚球藻和集胞藻（组I类1）将进行研究。 重点将是其独特的双组氨酸血红素协调和血红素翻译后修饰的决定因素。 (2)第二个目标承担的念珠藻血红蛋白（组I类2）的结构特征和后果，其高度疏水性血红素结合位点。 (3)在第三个目标中，血红素环境中的血红蛋白从肝螺杆菌（组III）将被描述，并与其独特的配体结合特性。 (4)最后，将设计新的NMR方法来探测每个蛋白质血红素口袋中关键氢键网络的复杂细节。 这四个具体的目标将有助于把这些有趣的蛋白质在血红蛋白超家族的背景下，促进其氨基酸序列的解释，并提供深入了解血红素化学和氧利用的新方面。更广泛的影响血红蛋白超家族是一个理想的主题，说明在生物化学领域的基本概念（代谢、调节）、化学（平衡、动力学和反应性）、生物学（进化）和物理学（光谱学）。 该项目为培训这些学科的研究生和本科生以及将研究成果纳入讲座和实验室提供了极好的机会。 一个新的生物物理课程，涵盖量子化学和光谱学将开发，将使用血红蛋白作为经常性的模型系统。 研究结果也将成为旨在提高研究生沟通技能的研讨会的基础。 系列讲习班将强调面向专业和更广泛的受众，从而促进参加讲习班的学生今后的外联活动。将通过出版物、参加会议、在网站上张贴和在公共数据库中存放结构数据来传播成果。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Hemoglobin is best known as the oxygen transporter responsible for the red color of blood. The hemoglobin superfamily of proteins, however, has members in all three domains of life, including bacteria. Little is known about bacterial hemoglobins, and it is expected that many participate in processes other than reversible oxygen binding. The project will explore the physico-chemical properties of bacterial hemoglobins belonging to the recently discovered 2/2 or "truncated" hemoglobin lineage of the superfamily. Of particular importance to the reactivity of heme proteins are the interactions involving the cofactor and the molecular motions that allow small ligands such as oxygen to reach the central iron. The goal of the research is therefore to establish robust relationships linking structure, dynamics, and reactivity. To this end, a combination of molecular biology approaches and spectroscopic methods (optical and nuclear magnetic resonance, NMR) will be applied to a set of proteins representative of two phylogenetically distinct groups of truncated hemoglobins (Group I and Group III) and two different classes within Group I. (1) In the first specific aim, the dynamic properties of the hemoglobins from the cyanobacteria Synechococcus and Synechocystis (Group I class 1) will be studied. The focus will be on the determinants of their distinctive bis-histidine heme coordination and heme post-translational modification. (2) The second aim bears on the structural characterization of a Nostoc punctiforme hemoglobin (Group I class 2) and the consequences of its highly hydrophobic heme binding site. (3) In the third aim, the heme environment in the hemoglobin from Helicobacter hepaticus (Group III) will be described and related to its unique ligand binding properties. (4) Finally, novel NMR methods will be devised to probe the intricate details of key hydrogen-bond networks in the heme pocket of each protein. The four specific aims will help place these intriguing proteins in the context of the hemoglobin superfamily, facilitate the interpretation of their amino acid sequences, and provide insight into novel aspects of heme chemistry and oxygen utilization.Broader ImpactsThe hemoglobin superfamily is an ideal subject with which to illustrate fundamental concepts in the fields of biochemistry (metabolism, regulation), chemistry (equilibria, kinetics and reactivity), biology (evolution), and physics (spectroscopy). The project offers excellent opportunities for the training of graduate and undergraduate students in these disciplines and for inclusion of research results in lectures and laboratories. A new biophysics course covering quantum chemistry and spectroscopy will be developed that will use hemoglobins as recurring model systems. The research results will also be at the basis of workshops designed to improve the communication skills of graduate students. The series will emphasize addressing both specialized and broader audiences, thereby facilitating future outreach activities of the students participating in the workshops. Results will be disseminated through publication, participation in conferences, posting on web sites, and deposition of structural data in public databases.