INVESTIGATING STRUCTURAL BASES OF SIGNAL TRANSDUCTION: PROTEIN ENG

我们正在研究信号转导的结构和功能 蛋白质，特别是异三聚体GTP结合蛋白(G 蛋白质)和G蛋白偶联受体(GPCRs)。当一种激动剂 如激素或神经递质结合其受体，交换 GDP的GTP与G蛋白结合是催化的。GTP绑定的Alpha G蛋白的亚基与β-伽马亚基分离 复杂，每一种都可以继续刺激下游 效应器。GTP被α亚基水解为GDP和 随后的杂三聚体重组关闭了该信号。而当 一些G蛋白亚基的结构已经在原子水平上被解决 受体的结构尚未确定。我们的特定 目标是通过设计来理解功能的结构基础 以及评估G蛋白和受体的突变形式。一 突变体类别包含两个或更多组氨酸，放置在 它们可以被金属离子连接起来。这样的联系可能会导致 依赖金属的蛋白质的激活或失活。在已知中 结构，这将增强我们对激活的知识 过程；在未知结构中，这也会产生距离 约束条件。另一类构造是融合到 含有四半胱氨酸基序的荧光蛋白或突变体 设计用于绑定小荧光分子闪光灯。这个 由此产生的物种发出的荧光将为我们提供信息 蛋白质的亚细胞定位和荧光共振 这些部分之间的能量转移(FRET)将显示出关联和 这些蛋白质在信号转导级联过程中解离。 CGL设施对本研究的许多步骤至关重要： 观察结晶学或模型化结构以设计突变体， 使突变体的属性合理化，并开发假设 关于蛋白质-配体和蛋白质-蛋白质相互作用，以及关于 激活和失活过程中的构象变化。

We are studying the structure and function of signal transduction proteins, specifically heterotrimeric GTP-binding proteins (G proteins) and G protein-coupled receptors (GPCRs). When an agonist such as a hormone or neurotransmitter binds its receptor, exchange of GTP for GDP bound to the G protein is catalyzed. The GTP-bound alpha subunit of the G protein separates from the beta-gamma subunit complex, and each of these can go on to stimulate downstream effectors. The hydrolysis of GTP to GDP by the alpha subunit and subsequent heterotrimer reassembly turns off the signal. While the structures of some G protein subunits have been solved, atomic-level structures have not been determined for the receptors. Our specific goals are to understand the structural basis of function by designing and evaluating mutant forms of the G proteins and receptors. One category of mutants contains two or more histidines placed so that they could be bridged by a metal ion. Such a link could cause metal-dependent activation or inactivation of the protein. In known structures, this would enhance our knowledge of the activation process; in unknown structures, this would also yield distance constraints. Another category of constructs are fusions to fluorescent proteins or mutants that contain a tetracysteine motif designed to bind the small fluorescent molecule FLASH. The fluorescence emitted by the resulting species will give us information on subcellular locations of the proteins, and fluorescence resonant energy transfer (FRET) between such moieties will show association and dissociation of these proteins during signal transduction cascades. The CGL facilities are crucial for many steps of this research: viewing crystallographic or modelled structures to design mutants, rationalizing the properties of the mutants, and to develop hypotheses about protein-ligand and protein-protein interactions, and about conformational changes involved in activation and deactivation.