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

总结： 标题：增强嗅觉受体的表达，用于气味的生化研究- 受体相互作用 我们的嗅觉是由嗅觉受体（ORs）介导的，这是最大的家族 G蛋白偶联受体（GPCRs）一些OR也在鼻子外部起作用， 协调重要的生理学; OR功能已被证明在肾脏中很重要， 皮肤、前列腺和多种癌组织中。尽管有突破性的进展， 描述了GPCR作用的结构基础，OR仍然是最重要的 在结构、配体结合和活化机制方面，未探索的GPCR类。 这主要是因为：1）异源细胞系中OR的低表达阻碍了 结构-功能研究和2）我们缺乏小分子，可以有效地激活， 抑制OR功能。在过去的两年里，Matsunami和Vaidehi发现了 氨基酸序列进化和结构特性，有助于OR 表情同时，Matsunami和Manglik使用了工程化的OR， 增强表达，以验证在洗涤剂中纯化OR的新方法， 结构和生物化学研究。在这些成功的基础上，我们建议 迭代预测检验中OR表达和配体发现的基本挑战 循环结合新的计算方法和实验测试有两个目标。 在目标1中，我们提出为六个人类OR设计突变OR， 用于体外研究的这些受体的过表达和用于大规模的 在洗涤剂中净化。在目标2中，我们将绘制OR配体结合位点并发现 新的高亲和力激动剂和拮抗剂， 配体对接和生化实验。我们还将制定方法， 通过冷冻电镜确定与气味剂和G蛋白结合的活性OR的结构。的 拟议工作的结果将为询问OR功能提供新的基础 和广泛可用的计算方法来加速OR领域。

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.