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，尽管它们具有副作用，包括呼吸抑制、便秘、成瘾和与我们患者的发病率和死亡率相关的其他药物，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β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。本研究将基于小鼠MUR的晶体结构，设计人μ受体的水溶性变体（wsMUR），研究其在N端和C端存在或缺失时的性质，并建立一个不需要放射性配体或哺乳动物细胞，直接研究阿片配体与受体相互作用的系统。本研究有两个主要目的。具体目标一是沿着我们最近发表的第一个工程受体，基于小鼠MUR的晶体结构设计和研究新一代wsMUR，并利用wsMUR和表面等离子体共振的优点，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中的大范围表面残基来计算工程化wsMURs的变体，以测试以下假设：1）存在使人MUR能够在大肠杆菌中以合理的水溶性和与其天然形式相当的性质表达的突变数目的最小要求。基于我们过去对膜蛋白进行蛋白质工程的经验，对跨膜部分的残基进行突变，以最大化水溶性并改善单体状态，用于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。该项目不仅为结构功能关系研究提供了wsMUR的各种变体，而且还产生了一个新的系统，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。该小组在工程化水溶性膜蛋白方面有着良好的记录。化学系的Saven教授是蛋白质工程专家，麻醉学系的刘教授是阿片受体专家。所有具体目标的初步数据都很有希望。

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.