Purification & Structural Analysis/Cannabinoid Receptor

描述(由申请人提供)： 大麻是最广泛使用的滥用药物之一，越来越多地被认为具有治疗作用。不幸的是，关于大麻受体CB1的高分辨率结构知识是有限的。这些知识将有助于化合物的开发，以更好地治疗成瘾、慢性疼痛和青光眼。 获得CB1结构信息的一个主要障碍是CB1是一种膜蛋白，因此很难获得足够数量的传统结构方法。此外，像G蛋白偶联受体家族的大多数其他成员一样，CB1对传统的纯化程序是困难的。 在CEBRA提案的目标I中，我们将为大量CB1的表达、溶解和纯化创造条件，为结晶研究奠定基础。在平行的轨道上，在AIM II中，我们将表达、纯化和结晶两个独立的CB1结构域，长的CB1 N端，以及截短形式的CBI的跨膜配体结合结构域。 最后，在目标III中，我们将评估CBI中细胞外环E-2的功能、结构和动力学。这些实验的目的是检验一个新的假设，即这个环在GPCRs的配体结合动力学和受体稳定性中起着普遍重要的作用。

DESCRIPTION (provided by applicant): Marijuana is one of the most widely used drugs of abuse, and is increasingly being ascribed therapeutic properties. Unfortunately, high-resolution structural knowledge about the marijuana receptor, called CB1, is limited. Such knowledge would aid in the development of compounds to better treat addiction, chronic pain and glaucoma. A major obstacle for obtaining CB1 structural information is the fact that CB1 is a membrane protein and thus difficult to obtain in quantities sufficient for traditional structural methods. Furthermore, like most other members of the G-protein coupled receptor family, CB1 is refractory to traditional purification procedures. In Aim I of this CEBRA proposal, we will establish conditions for expressing, solubilizing and purifying large amounts of CB1 to set the stage for crystallization studies. On a parallel track, in Aim II we will express, purify and crystallize two separate CB1 domains, the long CB1 N-terminus, and the transmembrane ligand-binding domain of a truncated form of CBI. Finally, in Aim III we will assess the function, structure and dynamics of extracellular loop E-2 in CBI. The goal of these experiments is to test the emerging hypothesis that this loop plays a universally important role in ligand binding kinetics and receptor stability in GPCRs.