Water soluble variants of the human mu opioid receptor

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，尽管其副作用包括呼吸抑制、便秘、成瘾和其他与患者发病率和死亡率相关的副作用，以及带来重大的经济和政治负担。由于镇痛和副作用都被认为是由受体（MUR）的激活引起的，因此更好地了解人类MUR的结构和功能关系将为解决这一临床困境提供线索。在本项目中，我们将基于新获得的小鼠mu受体晶体结构设计人类mu受体（wsMUR）的水溶性变体，研究该受体在N和C端存在或不存在时的特性，并建立一个研究阿片配体与受体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。这项研究有两个主要目的。在具体的目标1中，我们将基于小鼠MUR的晶体结构和我们最近发表的第一个工程受体版本来设计和研究新一代的wsMUR，并利用wsMUR和表面等离子体共振的优势建立一个研究阿片和受体直接相互作用的新系统。在具体目标2中，我们将通过改变跨膜区域的大范围表面残留物来计算工程wsmur的变体，以测试以下假设：1)人类MUR在大肠杆菌中的表达对突变数量的要求很低，并且具有合理的水溶性和与天然形式相当的性质~ 2)根据我们过去对膜蛋白进行蛋白质工程的经验，受体可以耐受35%的跨膜部分残基突变，以最大限度地提高水溶性并改善单体状态，以用于未来的高分辨率蛋白质动力学研究（即核磁）3)去除受体跨膜部分表面的半胱氨酸可以减少二硫键诱导的蛋白质二聚化。本项目不仅为结构功能关系研究提供了wsMUR的各种变体，而且还提供了一个可以研究阿片类药物与MUR直接相互作用的新系统，是一项重大的技术突破。因此，该项目旨在阐明MUR的分子机制，并有助于识别副作用更小、疼痛控制更好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。该小组在工程水溶性膜蛋白方面有着良好的记录。化学系的Saven教授是蛋白质工程方面的专家，麻醉系的Liu教授是阿片受体方面的专家。所有具体目标的初步数据都是有希望的。

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.