Design and Characterization of a Single-chain Hemoglobin

描述（由申请人提供）：已对珠蛋白家族的蛋白质进行了广泛研究，其中包括血红蛋白，血红蛋白作为潜在有用的携氧治疗剂，一直是许多研究和开发工作的重点。本研究的主要目的是设计和合成一种能可逆地协同结合氧的单链人血红蛋白。与多亚基复合物相比，单链血红蛋白可以更容易地设计为血液替代品，以获得最佳性能。单链血红蛋白分子的开发将是蛋白质工程的重大成就，也是氧气输送疗法发展的重要一步。单链血红蛋白也将允许在单个蛋白质框架中进行变构的高分辨率诱变研究，这将扩大我们对蛋白质功能中的协同性的理解。 拟议的研究将增加我们对蛋白质拓扑结构改变对蛋白质结构的影响的理解，以及在严格系统中的功能（即，变构蛋白）。我们的目标是展示改变复杂蛋白质中结构元件顺序的一般方法，从而扩大拓扑突变作为蛋白质工程工具的范围。 本研究的具体目标是：（1）以我们以前工作中描述的置换肌红蛋白为模型，制备环状置换的β-珠蛋白;（2）将置换的β-珠蛋白插入α-珠蛋白的G-H环中，形成单链α/β二聚体;（3）融合两个α/β二聚体，形成目标单链血红蛋白;（4）确定这些新构建体的功能（例如配体结合）性质和热力学稳定性。

DESCRIPTION (provided by applicant): The globin family of proteins has been extensively studied and includes hemoglobin which, as potentially useful oxygen carrying therapeutic, has been the focus of much research and development effort. The primary aim of this research is the design and synthesis of a single-chain human hemoglobin which binds oxygen reversibly and cooperatively. A single-chain hemoglobin could be more easily engineered for optimum performance as a blood substitute than the multisubunit complex. The development of a single-chain hemoglobin molecule would be a significant achievement in protein engineering as well as a significant step forward in the development of oxygen delivery therapeutics. A single-chain hemoglobin would also allow for high resolution mutagenesis studies of allostery in a single protein framework, which would expand our understanding of cooperativity in protein function. The proposed research will increase our understanding of the effects of altered protein topology on protein structure, and function in a stringent system (i.e., an allosteric protein). We aim to demonstrate general approaches for altering the order of structural elements in complex proteins, and thereby expand the scope of topological mutation as a protein engineering tool. The specific aims of the proposed research are: (1) to generate a circularly permuted beta-globin modeled on the permuted myoglobin characterized in our previous work; (2) to insert the permuted beta-globin into the G-H loop of alpha-globin to create single-chain alpha/beta dimers; (3) to fuse two alpha/beta dimers to create the target single-chain hemoglobin; (4) to determine the functional (e.g. ligand binding) properties, and thermodynamic stabilities of these novel constructs.