Novel variants of engineered water soluble mu opioid receptors with extensive mutations and removal of cysteines

Proteins. 2021 Oct;89(10):1386-1393. doi: 10.1002/prot.26160. Epub 2021 Jun 29.


We have shown that water-soluble variants of the human mu opioid receptor (wsMOR) containing a reduced number of hydrophobic residues at the lipid-facing residues of the transmembrane (TM) helices can be expressed in E. coli. In this study, we tested the consequences of increasing the number of mutations on the surface of the transmembrane domain on the receptor's aqueous solubility and ligand binding properties, along with mutation of 11 cysteine residues regardless of their solvent exposure value and location in the protein. We computationally engineered 10 different variants of MOR, and tested four of them for expression in E. coli. We found that all four variants were successfully expressed and could be purified in high quantities. The variants have alpha helical structural content similar to that of the native MOR, and they also display binding affinities for the MOR antagonist (naltrexone) similar to the wsMOR variants we engineered previously that contained many fewer mutations. Furthermore, for these full-length variants, the helical content remains unchanged over a wide range of pH values (pH 6 ~ 9). This study demonstrates the flexibility and robustness of the water-soluble MOR variants with respect to additional designed mutations in the TM domain and changes in pH, whereupon the protein's structural integrity and its ligand binding affinity are maintained. These variants of the full-length MOR with less hydrophobic surface residues and less cysteines can be obtained in large amounts from expression in E. coli and can serve as novel tools to investigate structure-function relationships of the receptor.

Keywords: G coupled protein receptor; computational protein design; human μ opioid receptor; opioid; water-soluble variant.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Escherichia coli / metabolism
  • Humans
  • Mutation
  • Receptors, Opioid, mu / chemistry*
  • Receptors, Opioid, mu / genetics
  • Solubility
  • Water


  • OPRM1 protein, human
  • Receptors, Opioid, mu
  • Water