Enhancing olfactory receptor expression for biochemical studies of odorant-receptor interactions

摘要： 标题：为气味的生化研究增加嗅觉受体的表达- 受体相互作用 我们的嗅觉是由嗅觉受体(ORs)调节的，嗅觉受体是最大的家族 G蛋白偶联受体(GPCRs)。一些OR也在鼻子外发挥作用 协调重要的生理；OR功能已被证明在肾脏中很重要， 皮肤、前列腺和多种癌症组织中。尽管取得了突破性的进展 勾勒出GPCR作用的结构基础，ORs仍然是最多的 未探索的类GPCRs在结构、配体结合和激活机制方面。 这主要是因为：1)异种细胞系中ORs的低表达阻碍了 结构-功能研究和2)我们缺乏能够有效地激活和 抑制或功能。在过去的两年里，松岛和怀德希发现了 与OR相关的氨基酸序列进化和结构特性 表情。同时，Matsunami和Manglik使用了工程OR 增强表达以验证净化洗涤剂中ORS的新方法 结构和生化研究。在这些成功的基础上，我们建议解决 迭代预测检验中OR表达和配体发现的基本挑战 循环结合了新的计算方法和实验测试，具有两个目标。 在目标1中，我们建议为六个人类ORs设计突变ORs，以使 这些受体在体外研究中的过表达及其在大规模研究中的可操作性 在洗涤剂中进行净化。在目标2中，我们将绘制OR配体结合位点图并发现 新的高亲和力激动剂和拮抗剂使用结构建模的组合， 配基对接和生化实验。我们还将开发方法来 用低温电子显微镜测定与气味和G蛋白结合的活性ORs的结构。这个 拟议工作的结果将为审问或功能提供新的基础 和广泛可用的计算方法来加速OR场。

Summary: Title: Enhancing olfactory receptor expression for biochemical studies of odorant- receptor interactions Our sense of smell is mediated by olfactory receptors (ORs), which are the largest family of G protein-coupled receptors (GPCRs). Some ORs also function outside the nose to coordinate important physiology; OR function has been shown to be important in kidney, skin, prostate, and in multiple cancer tissues. Despite groundbreaking advances in delineating the structural basis of GPCR action, ORs remain among the most unexplored class GPCRs in terms of structure, ligand binding and activation mechanism. This is primarily because: 1) low expression of ORs in heterologous cell lines impedes structure-function studies and 2) we lack small molecules that can potently activate and inhibit OR function. In the past two years, Matsunami and Vaidehi have uncovered amino acid sequence evolution and structural properties that contribute to OR expression. Simultaneously, Matsunami and Manglik used engineered ORs with enhanced expression to validate new approaches to purify ORs in detergents for structural and biochemical studies. Building on these successes, we propose to address fundamental challenges in OR expression and ligand discovery in iterative predict-test cycles combining novel computational methods and experimental testing with two aims. In Aim 1, we propose to engineer mutant ORs for six human ORs to enable overexpression of these receptors for in vitro studies and tractability for large-scale purification in detergents. In Aim 2, we will map OR ligand binding sites and discover new high affinity agonists and antagonists using a combination of structural modeling, ligand docking and biochemical experiments. We will also develop approaches to determine structures of active ORs bound to odorants and G proteins by cryo-EM. The outcome of the proposed work will provide new foundations to interrogate OR function and widely available computational approaches to accelerate the OR field.