Dynamical properties of G-protein coupled receptors using in-cell NMR

G.E. Folkers, I. Bodnariuc

Project description

Scientific background

G-protein coupled receptors are one of the largest and most diverse family of transmembrane proteins, with over 800 GPCRs identified in the human proteome. All GPCRs share a common 7- alpha helical transmembrane architecture. GPCRs transduce extracellular signals from small molecules, peptides, ions, and light to a diverse array of intracellular signaling pathways. Due to their large family diversity, physiological role and cellular localization in the plasma membrane, they are often targets for many therapeutics, with an estimation that 30% of available pharmaceutical drugs target a GPCR(1) .

Structure-based drug discovery has been used to identify new potential drug candidates for several GPCRs. However, structural and GPCR activation models are determined in biased systems due to the challenges of expressing and isolating receptors for in vitro studies. These biases include stabilizing mutations, reconstituting receptors in detergents or other membrane mimetics, and addition of nanobodies to anchor the receptor in a specific conformation(2) . There is increasing evidence that shows the membrane environment has significant influence on the receptor conformation and therapeutic output(3).

Research proposal

In-cell NMR and native-solid-state NMR offers the potential to study a full-length wildtype receptor in its natural membrane environment. By expressing the GPCR in mammalian cells and studying it in its native environment with solid-state NMR we can better understand its dynamics and conformational changes without bias.

Methods

  • Recombinant DNA technology including PCR, cloning, site directed mutagenesis, sequencing
  • Protein expression, purification and labelling of proteins in vitr0
  • Tissue culture
  • Transient transfection and electroporation of proteins
  • Light microscopy and immunofluorescence microscopy
  • Biophysical characterization of binding affinity using e.g. fluorescence anisotropy, thermophoresis

(1) Hauser, A. S.; Chavali, S.; Masuho, I.; Jahn, L. J.; Martemyanov, K. A.; Gloriam, D. E.; Babu, M. M. Pharmacogenomics of GPCR Drug Targets. Cell 2018, 172 (1-2), 41-54.e19. (acccessed 2024-10-10T09:56:48).

(2) Thal, D. M.; Vuckovic, Z.; Draper-Joyce, C. J.; Liang, Y. L.; Glukhova, A.; Christopoulos, A.; Sexton, P. M. Recent advances in the determination of G protein-coupled receptor structures. Curr Opin Struct Biol 2018, 51, 28-34. From NLM Medline.

(3) Christofidi, M.; Tzortzini, E.; Mavromoustakos, T.; Kolocouris, A. Effects of Membrane Cholesterol on the Structure and Function of Selected Class A GPCRs horizontal line Challenges and Future Perspectives. Biochemistry 2025. From NLM Publisher.