Crystallization of G-protein-Receptor Complexes

描述（由申请人提供）：G蛋白偶联受体（GPCR）是一种整合的膜蛋白，可调节多种不同的细胞信号传导途径，以控制人体的基本生理功能。信号生物化学中一个重要的未解决的问题是理解G蛋白被GPCR激活的机制。阐明激动剂活化状态的GPCR与其作用的G蛋白异源三聚体之间的复合物的三维结构对于解决该问题至关重要。因为在各种疾病状态中的药理学干预针对GPCR的特异性调节，所以在它们的活性信号传导状态下它们的三维结构的知识将提供指导受体激动剂和反向激动剂的设计的重要信息。该提议的目标是使用其中GPCR连接至G蛋白α亚基的融合蛋白作为支架，用于GPCR-G蛋白复合物在其活性、信号传导状态下的体内重建、过表达和纯化。将实施特定的载体设计和诱变策略以优化昆虫细胞系中稳定复合物的表达并使其能够有效纯化。将开发稳定GPCR-G蛋白复合物的方法，包括开发特异性去污剂/脂质组合和识别复合物天然结构的单克隆抗体FAB片段。将定义导致GPCR-G蛋白或其与单克隆FAB片段的复合物的晶体形成的条件，所述条件适合于确定其三维结构。由于其生物化学和药理学特性得到了很好的表征，β 2肾上腺素能受体-Gs融合蛋白将作为这些研究的原型。同时，将开发另外两种受体-G蛋白融合体，以利用这些受体、其配体和G蛋白信号传导伙伴的药理学和生物化学性质的变化，这可能提供特定的实验优势。

DESCRIPTION (provided by applicant): G Protein-Coupled Receptors (GPCRs) are integral membrane proteins that regulate multiple and diverse cellular signaling pathways to control essential physiological functions in humans. A significant unsolved problem in signaling biochemistry is to understand the mechanism by which G proteins are activated by GPCRs. Elucidation of the three-dimensional structure of the complex between a GPCR in its agonist activiated state with the G protein heterotrimer upon which it acts, is essential to the solution of this problem. Because pharmacological intervention in a variety of disease states is directed toward specific modulation of GPCRs, knowledge of their three-dimensional structures in their active signaling states will provide important information to guide the design of receptor agonists and inverse-agonists. The goal of this proposal is to use fusion proteins in which GPCRs are linked to G protein alpha subunits as scaffolds for in vivo reconstitution, over-expression and purification of GPCR-G protein complexes in their active, signaling state. Specific vector design and mutagenesis strategies will be implemented to optimize expression of stable complexes in insect cell lines and enable their efficient purification. Methods to stabilize GPCR-G protein complexes, including development of specific detergent/lipid combinations and FAB fragments of monoclonal antibodies that recognize the native structure of the complexes, will be developed. Conditions will the defined that lead to formation of crystals of GPCR-G proteins or their complexes with monoclonal FAB fragments, suitable for determination of their three-dimensional structures. Because its biochemical and pharmacological properties are well characterized, beta 2 adrenergic receptor-Gs fusion protein will serve as the prototype for these studies. At the same time, two other receptor-G protein fusions will be developed to exploit variations in the pharrmacological and biochemical properties of these receptors, their ligands and G protein signaling partners that may provide specific experimental advantages.