Characterization of a computationally designed water-soluble human μ-opioid receptor variant using available structural information.
Characterization of a computationally designed water-soluble human μ-opioid receptor variant using available structural information.
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DOI：
10.1097/aln.0000000000000308
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发表时间：
2014-10
期刊：
影响因子：
8.8
通讯作者：
Liu R
中科院分区：
文献类型：
--
作者：
Zhao X;Perez-Aguilar JM;Matsunaga F;Lerner M;Xi J;Selling B;Johnson AT Jr;Saven JG;Liu R
最近的鼠μ阿片受体（MUR）的X射线晶体结构使我们能够重新设计先前设计的人类MUR跨膜部分的水溶性变体（wsMUR-TM）。水溶性 MUR 的新变体 (wsMUR-TM_v2) 是根据鼠 MUR 晶体结构设计的。这种新的变体在大肠杆菌中表达并纯化。对该受体的特性进行了表征并与 wsMUR-TM 进行了比较。最初包含在 wsMUR-TM 设计中用于突变的七个残基被恢复到其天然身份。 wsMUR-TM_v2 包含总序列的 16% 突变。它被过表达并以高产率纯化。尽管观察到随着时间的推移形成二聚体和更高的寡聚体，但在两个月的时间内，wsMUR-TM_v2 在缓冲液中的浓度高达 7.5 mg/ml 时主要保持单体状态。其二级结构主要为螺旋状，与原始 wsMUR-TM 变体和天然 MUR 的二级结构相当。 wsMUR-TM_v2 对纳曲酮 (Kd ~ 70 nM) 的结合亲和力与 wsMUR-TM 非常一致。 wsMUR-TM_v2的螺旋含量随着温度的升高而降低，蔗糖的引入能够稳定蛋白质。基于鼠 MUR 晶体结构的信息，成功设计了一种仅具有 16% 突变的新型功能性 wsMUR-TM_v2，在大肠杆菌中表达并纯化。这不仅为溶液条件下的 MUR 研究提供了一种新颖的替代工具，而且还为蛋白质工程和结构功能关系提供了有价值的信息。
The recent X-ray crystal structure of the murine μ opioid receptor (MUR) allowed us to reengineer a previously designed water-soluble variant of the transmembrane portion of the human MUR (wsMUR-TM). The new variant of water soluble MUR (wsMUR-TM_v2) was engineered based upon the murine MUR crystal structure. This novel variant was expressed in E. coli and purified. The properties of the receptor were characterized and compared with those of wsMUR-TM. Seven residues originally included for mutation in the design of the wsMUR-TM, were reverted to their native identities. wsMUR-TM_v2 contains 16% mutations of the total sequence. It was overexpressed and purified with high yield. Although dimers and higher oligomers were observed to form over time, the wsMUR-TM_v2 stayed predominantly monomeric at concentrations as high as 7.5 mg/ml in buffer within a 2-month period. Its secondary structure was predominantly helical and comparable with those of both the original wsMUR-TM variant and the native MUR. The binding affinity of wsMUR-TM_v2 for naltrexone (Kd ~ 70 nM) was in close agreement with that for wsMUR-TM. The helical content of wsMUR-TM_v2 decreased cooperatively with increasing temperature, and the introduction of sucrose was able to stabilize the protein. A novel functional wsMUR-TM_v2 with only 16% mutations was successfully engineered, expressed in E. coli and purified based on information from the crystal structure of murine MUR. This not only provides a novel alternative tool for MUR studies in solution conditions, but also offers valuable information for protein engineering and structure function relationships.