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

增强嗅觉受体表达以进行气味受体相互作用的生化研究

基本信息

批准号：
10580041
负责人：
Aashish Manglik
金额：
$ 65.81万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-01 至 2027-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10580041
关键词：
Acceleration Address Affinity Agonist Amino Acid Sequence Back Binding Binding Sites Biochemical Biochemistry Biological Assay Carrier Proteins Cell Line Cell surface Cells Charge Chemicals Collaborations Communities Computing Methodologies Cryoelectron Microscopy Data Detergents Development Docking Engineering Event Evolution Family Family member Foundations G-Protein-Coupled Receptors GTP-Binding Proteins Goals Grant Human In Vitro Interdisciplinary Study Kidney Knowledge Ligand Binding Ligands Machine Learning Malignant Neoplasms Maps Mediating Modeling Molecular Nose Odorant Receptors Odors Olfactory Pathways Outcome Physiology Production Property Prostate Protein Family Protein Sequence Analysis Publishing Recombinants Role Signal Transduction Skin Smell Perception Structural Models Structure System Techniques Technology Testing Tissues Volatilization Work antagonist chemical binding design detection platform experience experimental study feeding improved innovation insight large scale production molecular modeling mutant novel novel strategies olfactory receptor overexpression predictive test protein purification receptor receptor binding receptor expression receptor function small molecule structural biology success thermostability tool

项目摘要

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.

概括：标题：增强嗅觉受体表达以进行气味剂的生化研究受体相互作用我们的嗅觉是由嗅觉受体 (OR) 介导的，它是最大的家族 G 蛋白偶联受体 (GPCR)。一些手术室也在鼻子外发挥作用协调重要的生理学； OR 功能已被证明对肾脏很重要，皮肤、前列腺和多种癌症组织中。尽管取得了突破性进展 OR 描述了 GPCR 作用的结构基础，仍然是最重要的在结构、配体结合和激活机制方面尚未探索的 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）我们缺乏可以有效激活和抑制 OR 功能。在过去的两年里，Matsunami 和 Vaidehi 发现了有助于 OR 的氨基酸序列进化和结构特性表达。同时，Matsunami 和 Manglik 使用了工程化 OR 增强表达以验证纯化洗涤剂中 OR 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测试中 OR 表达和配体发现的基本挑战结合新颖的计算方法和实验测试的循环有两个目标。在目标 1 中，我们建议为 6 个人类 OR 设计突变 OR，以实现这些受体的过度表达用于体外研究和大规模的易处理性洗涤剂中的净化。在目标 2 中，我们将绘制 OR 配体结合位点并发现结合结构建模的新型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。这拟议工作的结果将为询问 OR 功能提供新的基础以及广泛可用的计算方法来加速 OR 领域。