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

CELL-FREE EXPRESSION TO INVESTIGATE SITE-SPECIFIC DYNAMICS OF GPCRs

Not scheduled
4h
Harnack House and Henry Ford Building

Harnack House and Henry Ford Building

Board: 244
Poster Posters

Speaker

Dr Ulrike Krug (Institute for Medical Physics and Biophysics, Leipzig University, Leipzig, Germany)

Description

The high relevance of G protein-coupled receptors (GPCRs) becomes apparent from their diverse distribution in human tissue and their participation in a variety of physiological functions. This makes them highly interesting molecules for basic research and also a major target for pharmaceutical drugs.
Besides the structural architecture of the seven transmembrane proteins and its interaction with ligands and intracellular binding partners, their dynamic properties represent a fundamental role for their function in signal transduction. Recently, we were able to get insight into the dynamics of peptide-binding class A GPCRs by solid-state NMR spectroscopy. It was possible to characterize the dynamics of the receptors in the presence and in the absence of different ligands and embedded in different membranes [1,2,3]. The order parameters that were derived from the Dipshift experiments gave a measure for the dynamic properties of the receptors. However, they were determined for the fully 13C labeled neuropeptide Y and growth hormone secretagogue receptors, and thus represent average values of all residues. They did not resolve any specific properties of distinct sites. Here we show how to use cell-free expression for selective isotopic labelling of GPCRs. The receptors are expressed in the precipitated form and are reconstituted in lipid bicelles. The resulting NMR spectra show a reduced spectral complexity and allow the investigation of specific amino acid sites, e.g. with respect to receptor activation.

References:
1.) Schmidt, P., Thomas, L., Müller, P., Scheidt, H. A., & Huster, D., Chemistry, 2014, 20(17), 4986-4992.
2.) Thomas, L., Kahr, J., Schmidt, P., Krug, U., Scheidt, H. A., & Huster, D. J. Biomol.- NMR, 2015, 61(3-4), 347-359.
3.) Schrottke, S., Kaiser, A., Vortmeier, G., Els-Heindl, S., Worm, D., Bosse, M., Schmidt, P., Scheidt, H.A., Beck-Sickinger, A.G., & Huster, D., Sci. Rep., 2017, 7, 46128.

Primary authors

Dr Ulrike Krug (Institute for Medical Physics and Biophysics, Leipzig University, Leipzig, Germany) Dr Peter Schmidt (Institute for Medical Physics and Biophysics, Leipzig University, Leipzig, Germany) Prof. Daniel Huster (Institute for Medical Physics and Biophysics, Leipzig University, Leipzig, Germany)

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