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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. coliand 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.
This is a non-final version of an article published in final form in Anesthesiology. 2014 October ; 121(4): 866–875. doi:10.1097/ALN.0000000000000308
Zhao, X., Perez Aguilar, J., Matsunaga, F., Lerner, M., Xi, J., Selling, B., Johnson, A. C., Saven, J. G., & Liu, R. (2014). Characterization of a Computationally Designed Water-Soluble Human μ Opioid Receptor Variant Using X-ray Structural Information. Anesthesiology, 121 (4), 866-875. http://dx.doi.org/10.1097/ALN.0000000000000308
Date Posted: 07 December 2016