Resolving Conformational Changes Within the Mu-opioid receptor via smFRET and its Correlation with G-Protein Activation
Abstract/Contents
- Abstract
- The Mu-opioid receptor is a therapeutic target of great importance and underlies the site of action for opiates. These opiates can have adverse effects that are related to the ability of the Mu-opioid receptor to signal with a defined level of activity. Alternatively, opiates may cause a particular bias to produce favorable therapeutic goals such as analgesia and avoid side-effects such as respiratory depression that may lead to death. Here, we have purified and labelled the Mu-opioid receptor to study the change in conformation it undergoes when binding opiates. Conformational changes were measured via single molecule FRET (smFRET), a biophysical technique that reveals the functions, kinetics, and conformation dynamics of individual molecules by monitoring the energy transfer between two probes (i.e., a donor and acceptor probe). We identified ligand dependent changes in conformational states in real time and directly measured the conformational changes that the receptor undergoes when it interacts with its cognate G-protein. Additionally, we attempted to reinsert purified Mu-opioid receptor by nanopore electroporation into live cells to confirm its functionality in the cell membrane. This attempt laid the groundwork for an in vivo model required for future evaluation of ligand driven conformational effects. Continued work will use more sophisticated methods of single-molecule FRET to resolve the conformational states of the receptor and to characterize the effects that ligands of varying efficacies have on G-protein interactions.
Description
| Type of resource | text |
|---|---|
| Date modified | December 5, 2022 |
| Publication date | May 5, 2022; May 2022 |
Creators/Contributors
| Author | Bowen, Willow |
|---|---|
| Thesis advisor | Chu, Steven |
| Thesis advisor | Barnes, Christopher |
| Degree granting institution | Stanford University, Department of Biology |
Subjects
| Subject | Biology |
|---|---|
| Subject | single molecule Fluorescent Resonance Energy Transfer (smFRET) |
| Subject | Mu-opioid receptor (µOR) |
| Subject | G-protein Coupled Receptor (GPCR) |
| Subject | Nanopore Electroporation |
| Genre | Text |
| Genre | Thesis |
Bibliographic information
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- License
- This work is licensed under a Creative Commons Attribution Non Commercial 4.0 International license (CC BY-NC).
Preferred citation
- Preferred citation
- Bowen, W. and Chu, S. (2022). Resolving Conformational Changes Within the Mu-opioid receptor via smFRET and its Correlation with G-Protein Activation. Stanford Digital Repository. Available at https://purl.stanford.edu/nw387mx9436
Collection
Undergraduate Theses, Department of Biology, 2021-2022
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- wtree99@stanford.edu
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