Bridging Soluble Variants and Native-like Environments to Probe Mu-Opioid Receptor Structure and Dynamics
Date Issued
August 2026
Author(s)
Agyemang, Eugene Boateng
The University of Tennessee
Advisor(s)
Rajan Lamichhane
Additional Advisor(s)
Elias Fernandes
Francisco Barrera
Naresh Osti
Abstract
Opioid analgesics are indispensable for the treatment of moderate-to-severe pain and act primarily through the mu-opioid receptor (MOR), a G protein-coupled receptor (GPCR). However, biophysical studies of MOR are challenging due to its membrane localization, low expression, and poor solubility. In this work, we used engineered MOR platforms to overcome these barriers and characterize receptor structure and dynamics in solution and native-like environments. An engineered water-soluble MOR variant (wsMOR) was used to determine whether key receptor properties could be preserved outside the lipid bilayer. Despite extensive redesign, wsMOR retained major structural and functional features, including α-helical secondary structure, thermal stability, ligand binding, and a largely monomeric state in solution, establishing that essential features of MOR can be maintained outside the membrane in an engineered soluble form. Receptor dynamics were then examined across picosecond-to-millisecond timescales using quasi-elastic neutron scattering (QENS) and single-molecule Förster resonance energy transfer (smFRET). These studies revealed that ligand binding reshapes the MOR conformational landscape by restricting fast internal motions and redistributing receptor populations among four distinct conformational states, linking local molecular motions to larger-scale conformational transitions associated with receptor activation. To restore membrane context, a single-molecule method was developed using styrene-maleic acid lipid particles, unnatural amino acid incorporation, and site-specific fluorescent labeling. This approach enabled the isolation and labeling of MOR in native membrane-derived nanoparticles and provides a foundation for future studies of receptor behavior in more physiologically relevant settings. Taken together, these studies show that engineered receptor systems can make MOR experimentally tractable while preserving key structural, functional, and dynamic properties of the receptor. Beyond MOR, the methods developed here expand the toolkit for investigating GPCR structure, dynamics, and organization across solution and membrane environments, creating new opportunities for mechanistic studies, ligand discovery, and tool development in GPCR biology.
Degree
Doctor of Philosophy
Major
Life Sciences
Embargo Date
August 1, 2027