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，特别是异源三聚体GTP结合蛋白（G 蛋白质）和G蛋白偶联受体（GPCR）。 当激动剂 如激素或神经递质结合其受体， 与G蛋白结合的GDP的GTP被催化。 GTP结合的alpha G蛋白的亚基与β-γ亚基分离 每一个都可以刺激下游 效应器 GTP通过α亚基水解为GDP， 随后的异源三聚体重组关闭了信号。 而 一些G蛋白亚基的结构已经在原子水平上得到了解决， 受体的结构尚未确定。 我们的具体 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 一类突变体含有两个或多个组氨酸， 它们可以通过金属离子桥接。 这种联系可能会导致 蛋白质的金属依赖性活化或失活。 以已知 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 另一类构建体是与 含有四半胱氨酸基序的荧光蛋白或突变体 设计用来结合小的荧光分子FLASH。 的 产生的物质发出的荧光会给我们提供信息 在蛋白质的亚细胞位置上， 这些部分之间的能量转移（FRET）将显示缔合， 在信号转导级联过程中这些蛋白质的解离。 CGL设施对于本研究的许多步骤至关重要： 观察晶体学或模型结构来设计突变体， 合理化突变体的性质，并发展假设 蛋白质与配体和蛋白质与蛋白质的相互作用， 构象变化参与激活和失活。

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.