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：
10465009
负责人：
Aashish Manglik
金额：
$ 68.86万
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/10465009
关键词：
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 Electron Microscopy Engineering Event Evolution Family Family member Foundations G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs 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 Work antagonist base chemical binding cryogenics design 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：增强气味生化研究的嗅觉受体表达受体相互作用我们的嗅觉是由嗅觉受体（OR）介导的，这是最大的家庭 G蛋白偶联受体（GPCR）的有些OR在鼻子外也起作用协调重要的生理；或功能已显示在肾脏中很重要，皮肤，前列腺和多种癌组织。尽管有开创性的进步描述GPCR行动的结构性基础，ORS仍然是最多的在结构，配体结合和激活机制方面未开发的GPCR类。这主要是因为：1）异源细胞系中ORS的低表达阻碍结构功能研究和2）我们缺乏可以有效激活和的小分子抑制或功能。在过去的两年中，Matsunami和Vaidehi发现了氨基酸序列的演化和结构特性，有助于或表达。同时，Matsunami和Manglik使用了工程设计的ORS 增强的表达以验证新方法净化清洁剂中的ORS 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的基本挑战或表达和配体发现将新型计算方法和实验测试与两个目标结合的循环。在AIM 1中，我们建议为六个人类ORS设计突变体以启用这些受体的过表达用于体外研究和大规模的障碍性洗涤剂中的纯化。在AIM 2中，我们将映射或配体绑定位点并发现新的高亲和力激动剂和拮抗剂，结合结构建模的组合，配体对接和生化实验。我们还将开发方法确定通过冷冻EM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或功能提供新的基础并广泛可用的计算方法来加速或字段。