Water soluble variants of the human mu opioid receptor

人类μ阿片受体的水溶性变体

• 批准号: 8749007
• 负责人: Renyu Liu
• 金额: $ 31.69万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-05 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8749007
• 关键词: Achievement; Address; Adverse effects; Analgesics; Anesthesiology; Back; Binding; Biochemical; Biological Assay; Cessation of life; Chemistry; Clinical; Constipation; Cysteine; Data; Dimerization; Economics; Engineering; Escherichia coli; Excision; Future; G-Protein-Coupled Receptors; Generations; Homology Modeling; Human; Length; Ligands; Mammalian Cell; Membrane; Membrane Proteins; Molecular; Morbidity - disease rate; Mus; Mutation; Nuclear Magnetic Resonance; Opioid; Opioid Analgesics; Opioid Receptor; Pain management; Patients; Perioperative; Pharmaceutical Preparations; Production; Property; Protein Dynamics; Protein Engineering; Proteins; Public Health; Publishing; Radioactive; Receptor Activation; Resolution; Role; Solubility; Solutions; Specific qualifier value; Structure; Structure-Activity Relationship; Surface; Surface Plasmon Resonance; System; Technology; Testing; Transmembrane Domain; United States; Variant; Ventilatory Depression; Water; addiction; base; chronic pain; design; disulfide bond; experience; improved; mortality; mu opioid receptors; novel; novel strategies; prevent; professor; protein function; public health relevance; receptor; receptor expression; receptor structure function; thermostability; tool; water solubility

DESCRIPTION (provided by applicant): Opioids remain the major medication for perioperative and chronic pain management despite their side effects, which include respiratory depression, constipation, addiction and others related to morbidity and mortality in our patients, as well as presenting a significant economic and political burden. Since both the analgesic and side effects are thought to arise from activation of the ¿-receptor (MUR), a better understanding of the structural and functional relationships of human MUR will provide clues in addressing this clinical dilemma. In this project, we will engineer water soluble variants of the human mu receptor (wsMUR) based on the newly available crystal structure of murine MUR, investigate the properties of the receptor in the presence or absence of N and C terminus, and establish a system in investigating the direct interaction of an opioid ligand with the receptor without needing radioactive ligands or mammalian cells. There are 2 major aims for this study. In specific aim 1, we will engineer and study a new generation of wsMUR based on the crystal structure of murine MUR along with our first version of engineered receptor published recently and establish a novel system in investigating the direct interaction of opioid and receptor by taking the advantages of wsMUR and surface plasmon resonance. In specific aim 2, we will computationally engineer variants of wsMURs by changing wide ranges of surface residues in the transmembrane region to test the following hypotheses: 1) There is a minimal requirement of the number of mutations enabling the human MUR to be expressed in E coli with a reasonable water solubility and comparable properties to its native form~ 2) The receptor may tolerate 35% of the mutations for the residues in the transmembrane portion based on our past experiences of protein engineering on the membrane proteins to maximize water solubility and improve monomeric states for future high resolution protein dynamics studies (i.e. nuclear magnetic resonance) in solution conditions~ 3) Removal of cysteine on the surface of the transmembrane portion of the receptor could reduce disulfide bond induced protein dimerization. This project not only provides various variants of wsMUR for structure function relationship studies, but also yields a novel system that can investigate the direct interaction between opioids and MUR, and offer a significant technology breakthrough. Thus, this project stands to elucidate the molecular mechanisms of MUR and aid in the identification of novel opioid analgesics with reduced side effects, improved pain control, and address issues related to opioid administration and abuse. This group has a track record in engineering water soluble membrane proteins. Professor Saven at the Department of Chemistry is an expert on protein engineering and Professor Liu at the Department of Anesthesiology is an expert on opioid receptors. The preliminary data for all the specific aims are promising.

描述（由申请人提供）：阿片类药物仍然是围手术期和慢性疼痛管理的主要药物，尽管它们有副作用，包括呼吸抑制、便秘、成瘾以及与患者发病率和死亡率相关的其他副作用，并且带来了显着的经济和经济效益。因为镇痛作用和副作用都被认为是由 ¿ 的激活引起的。 -受体（MUR），更好地了解人类 MUR 的结构和功能关系将为解决这一临床困境提供线索。在这个项目中，我们将基于新近获得的人类 mu 受体（wsMUR）的水溶性变体。小鼠MUR的晶体结构，研究在N和C末端存在或不存在的情况下受体的性质，并建立研究阿片类配体与受体的直接相互作用的系统，而不需要放射性配体或哺乳动物细胞。这项研究的 2 个主要目标 在具体目标 1 中，我们将基于鼠 MUR 的晶体结构以及我们最近发表的第一个工程受体来设计和研究新一代 wsMUR，并建立一个研究直接相互作用的新系统。利用 wsMUR 和表面等离子共振的优势，我们将通过计算设计 wsMUR 的变体，通过改变跨膜区域的广泛表面残基来测试以下假设： 1) 突变数量的最低要求使人类 MUR 能够在大肠杆菌中表达，并具有合理的水溶性和与其天然形式相当的特性~ 2) 受体可以耐受 35% 的残基突变跨膜部分基于我们过去对膜蛋白进行蛋白质工程的经验，以最大限度地提高水溶性并改善单体状态，以用于未来溶液条件下的高分辨率蛋白质动力学研究（即核磁共振）〜3）半胱氨酸的去除该项目不仅为结构功能关系研究提供了wsMUR的各种变体，而且还产生了一个可以研究阿片类药物和MUR之间直接相互作用的新系统。因此，该项目旨在阐明 MUR 的分子机制，并帮助鉴定具有减少副作用、改善疼痛控制的新型阿片类镇痛药，并解决与阿片类药物给药和滥用相关的问题。该小组在水溶性膜蛋白工程方面拥有丰富的经验，化学系的 Saven 教授是蛋白质工程方面的专家，麻醉学系的 Liu 教授是阿片类受体方面的专家。所有具体目标的初步数据如下。有前途。