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

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.

DEER Analysis of GPCR Conformational Heterogeneity

“…Both MTSL and IAP consistently and stably derivatized the native cysteines as well as the engineered cysteine giving rise to complex distance distributions in DEER. Similar behavior was recently observed in preliminary DEER data on the Y2 receptor [ 40 ].…”

Neuropeptide Y Reduces Nasal Epithelial T2R Bitter Taste Receptor–Stimulated Nitric Oxide Production

“…We observe effects of NPY on T2R-induced NO responses as low as 1 nM. The pKd of recombinant NPY2R vs I125-NPY was reported to be~10.17 (~0.068 nM) [95] and EC 50 of~0.3 nM in a heterologous expression system [96]. The apparent EC 50 for NPY is likely higher than the binding of NPY to its receptor due to downstream nonlinearity.…”

Towards Probing Conformational States of Y2 Receptor Using Hyperpolarized 129Xe NMR

“…We introduced an additional cysteine at position 202 (Y2R-A202C) directly neighboring the native C203 in ECL2. The Y2R-A202C mutant was used in previous EPR studies, and wild-type-like behavior was confirmed [ 22 ].…”

“…Specifically, the determination of the concentration of the conjugate in its different states, i.e., the fractional populations, could be very helpful in the exploration of receptor activity. Along this route, a number of varied experimental schemes may be applied, e.g., using construct architectures of different linker length or linker flexibility in Y2R activation by different ligands, individually or in combination, at the extra- and intracellular receptor binding sites [ 22 ] to ultimately arrive at a quantitative and comprehensive description of Y2R activity. Moreover, complementary solution- or solid-state NMR spectroscopy using specific isotopic labeling may be undertaken to verify contact sites and MD employed for refining the insights into Y2R structural dynamics.…”

“…The functional reconstitution of the receptor into non-isotropic DMPC bicelles was performed as previously described [ 24 ]. CPM and fluorescence polarization assays were performed as previously conducted [ 22 ]. After functional reconstitution, Y2R-A202C was incubated with 10× molar excess of CrA-Cy3-mal overnight at 4 °C and pH 7 to form Y2R-A202C-CrA.…”