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（通过应用程序提供）：尽管副作用，包括呼吸抑郁症，便秘，成瘾以及与我们患者的发病率和死亡率有关的其他副作用，但仍具有巨大的经济和政治负担，但仍是定期和慢性疼痛管理的主要药物。由于镇痛和副作用均被认为是由受体（MUR）的激活引起的，因此对人MUR的结构和功能关系的更好理解将为解决这一临床困境提供线索。在这个项目中，我们将根据Murine Mur的新晶体结构来设计人类MU受体（WSMUR）的水固体变体，研究在存在或不存在N和C末端的情况下，研究受体的性质，并建立一个系统，以调查不需要放射性ligandiel ligands或mamalalian或mammalian或mammalalian或mammalmalian或mammalmalian或mammalmalmalian或mammalmalian。这项研究有2个主要目标。在特定的目标1中，我们将根据鼠MUR的晶体结构以及我们最近出版的第一版工程接收器进行设计和研究新一代WSMUR，并建立了一种新型系统，以研究阿片类药物和接收器的直接相互作用，通过获取WSMUR和Surface Plasmon Resonance的优势。在特定目标2中，我们将通过更改跨膜区域中的宽范围来测试以下假设来计算WSMURS的计算工程师变体：1）对突变的数量的需求最少，使人类的MUR数量能够在E Coli中以合理的水性和可比性的方式表达，以便于其本机溶解的繁殖物，而在其本机上可以进行繁殖35）。跨膜部分基于我们过去对膜蛋白上蛋白质工程的经验，以最大程度地溶于水并改善单体状态，以使未来的高分辨率蛋白质动力学研究（即核磁共振）在溶液条件下〜3）去除在受体跨内膜部分的跨膜部分上的半胱氨酸的去除量可以降低二胰蛋白酶构造的蛋白质化。该项目不仅为结构功能关系研究提供了各种WSMUR的变体，而且还产生了一个新型系统，可以研究阿片类药物与MUR之间的直接相互作用，并提供了重要的技术突破。这是该项目旨在阐明MUR的分子机制，并有助于鉴定新的阿片类镇痛药，并减少副作用，改善疼痛控制以及解决与阿片类药物给药和滥用有关的问题。该组在工程水固体膜蛋白方面具有往绩记录。化学系的Saven教授是蛋白质工程专家，麻醉学系的Liu教授是阿片受体的专家。承诺所有特定目标的初步数据。