Design and Characterization of a Single-chain Hemoglobin

描述(申请人提供)：珠蛋白家族的蛋白质已被广泛研究，包括作为潜在有用的携氧治疗的血红蛋白，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与氧可逆、协同结合的单链人血红蛋白。单链血红蛋白可以比多亚单位复合体更容易被设计成作为血液替代品的最佳性能。单链血红蛋白分子的开发将是蛋白质工程的一项重大成就，也是氧气输送疗法发展的重要一步。单链血红蛋白还可以在单一的蛋白质框架内对变构进行高分辨率的突变研究，这将扩大我们对蛋白质功能中的协同作用的理解。 这项拟议的研究将增加我们对蛋白质拓扑结构变化对蛋白质结构的影响的理解，以及在严格的系统(即变构蛋白质)中的功能。我们的目标是展示改变复杂蛋白质结构元素顺序的一般方法，从而扩大拓扑突变作为蛋白质工程工具的范围。 拟议研究的具体目标是：(1)以我们先前工作中描述的置换的肌红蛋白为模型生成循环置换的β-珠蛋白；(2)将置换的β-珠蛋白插入到α-珠蛋白的G-H环中以创建单链的α/β二聚体；(3)融合两个α/β二聚体以创建靶向单链血红蛋白；(4)确定这些新结构的功能性质(例如配体结合)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.