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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.

简介: