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

Molecules

Anette Kaiser¹  AnnetteG. Beck-Sickinger¹  HalehH. Haeri²  Dariush Hinderberger²  JeannetteM. Laugwitz³  Ulrike Krug³  Peter Schmidt³ 

[1] Faculty of Life Sciences, Institute of Biochemistry, University of Leipzig, Bruederstrasse 34, 04103 Leipzig, Germany;Institute of Chemistry, Martin-Luther-University of Halle-Wittenberg, Von-Danckelmann-Platz 4, 06120 Halle (Saale), Germany;Medical Faculty, Institute of Medical Physics and Biophysics, University of Leipzig, Haertelstasse 16-18, 04107 Leipzig, Germany;

关键词: GPCR;    Y2R;    EPR;    DEER;    refolding;    nitroxide spin labels;   

DOI  :  10.3390/molecules25184143

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown