25-30 August 2019
Henry Ford Building
Europe/Berlin timezone

Conformational dynamics of a wildtype and an evolved G protein-coupled receptor

Not scheduled
Harnack House and Henry Ford Building

Harnack House and Henry Ford Building

Board: 606
Poster-Late Late-Breaking Posters


Prof. Joshua Ziarek (Indiana University)


G protein-coupled receptors (GPCRs) are membrane-integrated signaling proteins that translate extracellular stimuli into an intracellular response for processes ranging from vision, smell and taste to immunologic, neurologic and reproductive functions. Only 16% of the superfamily are clinically targeted – and yet these comprise nearly 35% of all marketed drugs. Despite their obvious therapeutic potential, the molecular details of signal transduction remain largely unknown. GPCRs are notoriously difficult to study due to their expansive conformational landscape and dynamic nature, which substantially reduces their long-term stability for biochemical, structural, and pharmacological studies. Although their intrinsic dynamics make them intriguing targets for solution-state measurements, instability and poor prokaryotic expression has limited studies to a small number of NMR-active probes (15N-Val, 15N-Met, 19F-BTFMA). Applying directed evolution to the neurotensin receptor, we produced a functional construct (enNTS1) with enhanced bacterial expression, detergent and thermal stability. Most importantly, E. coli expression opens the door to robust uniform and selective isotope labeling strategies in a perdeuterated background. Using 19F-NMR we are currently comparing the conformational dynamics of our evolved en2NTS1 construct to wildtype NTS1. We have collected spectra of both proteins in the apo form as well as the antagonist, agonist and G protein-bound states. Our preliminary data suggests that en2NTS1 can occupy equivalent conformational space but with slower exchange kinetics. Interesting, receptor evolution introduced mutations in key functional areas such as the toggle switch region and sodium ion binding site, but retained functional activity at ~75% wildtype. We are exploring the effect of these amino acid substitution on receptor function, structure and dynamics using aromatic stereo-array isotope label (SAIL) and 13C-methyl labeled receptor. Finally, we’re applying real-time NMR techniques to monitor phosphorylation of intracellular sites following receptor activation. Taken together, we aim to draw a complete picture of GPCR activation and signaling in solution.

Primary authors

Mr Austin Dixon (Indiana University) Dr Fabian Bumbak (Indiana University) Dr Daniel Scott (Florey Institute; University of Melbourne) Prof. Masatsune Kainosho (Tokyo Metropolitan University) Prof. Joshua Ziarek (Indiana University)

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