Understanding the Conformational Dynamics of the Glucagon Receptor in a Native Membrane Environment Using Single-Molecule FRET

Author

Susmita Khanal, University of Tennessee, KnoxvilleFollow

Date of Award

8-2024

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Rajan Lamichhane

Committee Members

Amit Joshi, Barry Bruce

Abstract

G Protein-coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to most hormones, metabolites, cytokines, and neurotransmitters. Representing the most prominent family of proteins with approximately 800 members in humans, GPCRs are the target for more than 40% of current pharmaceutical drugs. The structural and dynamic information is vital to understanding the activation mechanism of these receptors. Even if the structures of GPCRs at different conformational states are available, the dynamics of those conformational states at the molecular level are still missing. Thus, we used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the Transmembrane Domain VI (TMD6) of the glucagon receptor (GCGR), a class B family GPCR involved in glucose homeostasis. We expressed GCGR in mammalian cells, extracted and purified them in the native environment using Styrene-maleic Acid Lipid Particles (SMALPs), and analyzed them at a single-molecule level. We used Single-molecule Förster Resonance Energy Transfer (smFRET) to observe the conformational dynamics of the donor and acceptor fluorophore-labeled GCGR molecules in SMALPs. We observed the highly dynamic behavior of the transmembrane domain VI of GCGR-SMALPs. Upon addition of full agonist glucagon, significant alteration in the dynamics was observed, as evident by a shift towards the lower FRET intermediate state. However, the addition of an antagonist, NNC0640, restricted the flexibility of the receptor TMD6. Additionally, in the presence of mini-Gs, we observed a significant population of the receptor in low-FRET active and mid-FRET, intermediate state. These results provide new insight into the structural dynamics of glucagon receptor and receptor activation, which might be helpful for diabetes drug design in the future.

Recommended Citation

Khanal, Susmita, "Understanding the Conformational Dynamics of the Glucagon Receptor in a Native Membrane Environment Using Single-Molecule FRET. " Master's Thesis, University of Tennessee, 2024.
https://trace.tennessee.edu/utk_gradthes/11796