Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements

Jeannette M Laugwitz,Anette Kaiser,Annette G Beck-Sickinger,Peter Schmidt,Dariush Hinderberger,Haleh H Haeri,Ulrike Krug

doi:10.3390/molecules25184143

Jeannette M Laugwitz, Anette Kaiser + Show 5 more

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https://doi.org/10.3390/molecules25184143

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Abstract

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor.

Highlights

G protein-coupled receptors (GPCRs) play a central role in numerous signal transduction pathways across the cell membrane, which are initiated by binding of extracellular ligands to the receptor and subsequent activation of different intracellular signaling pathways
In addition to a comparable activity of this variant with thethe wild-type receptor, it isbridge characterized by increased stability which physiological activity of this variant with the wild-type receptor, it is characterized by increased facilitates folding into its native conformation during functional reconstitution [34]
The results showed thatthree the water-exposed conformation be attributed to an immobile component, having dynamics of about times slower than the second could be attributed to an immobile component, having dynamics of about three times conformation

Summary

Introduction

G protein-coupled receptors (GPCRs) play a central role in numerous signal transduction pathways across the cell membrane, which are initiated by binding of extracellular ligands to the receptor and subsequent activation of different intracellular signaling pathways. Due to this central role in various biochemical signal transduction cascades, GPCRs are of high pharmacological relevance [1]. One major focus in current GPCR research deals with the detailed molecular understanding of the ligand-receptor interaction in terms of structural dynamics features and binding routes. The inherent limitation of crystal structures, as they provide only static snapshots of the molecules in a non-membrane environment of artificially stabilized GPCRs [4], are increasingly compensated by other biophysical methods such as cryo-electron microscopy [5,6], nuclear magnetic resonance (NMR) [7,8,9,10,11], or electron paramagnetic resonance (EPR) [12,13] spectroscopy

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