The dynamics of the G protein-coupled neuropeptide Y2 receptor in monounsaturated membranes investigated by solid-state NMR spectroscopy

Abstract

In contrast to the static snapshots provided by protein crystallography, G protein-coupled receptors constitute a group of proteins with highly dynamic properties, which are required in the receptors' function as signaling molecule. Here, the human neuropeptide Y2 receptor was reconstituted into a model membrane composed of monounsaturated phospholipids and solid-state NMR was used to characterize its dynamics. Qualitative static (15)N NMR spectra and quantitative determination of (1)H-(13)C order parameters through measurement of the (1)H-(13)C dipolar couplings of the CH, CH2 and CH3 groups revealed axially symmetric motions of the whole molecule in the membrane and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (S(backbone) = 0.59-0.67, S(CH2) = 0.41-0.51 and S(CH3) = 0.22) obtained in directly polarized (13)C NMR experiments demonstrate that the Y2 receptor is highly mobile in the native-like membrane. Interestingly, according to these results the receptor was found to be slightly more rigid in the membranes formed by the monounsaturated phospholipids than by saturated phospholipids as investigated previously. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, the incorporation of cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine into the membrane did not have a significant influence on the molecular mobility of the Y2 receptor.

Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes

“…Axially symmetric rotational diffusion of the membrane protein is manifested by anisotropic lineshapes indicative of axially symmetric chemical shift anisotropy tensors (η = 0). Superimposed to these broad lineshapes, large amplitude motions give rise to very narrow lines detected at the isotropic chemical shift of the respective resonances2224. However, as static 15 N NMR spectra are almost always acquired using cross polarization (CP), the recorded spectra show a strong bias towards rigid structures and dynamic features are only detected if sufficiently long CP contact times are used24.…”

“…This observation indicates that in addition to the axially symmetric rotational diffusion of the GHS receptor in the membrane, significant portions of the molecule also undergo large amplitude motions. We have previously quantified the proportion of highly mobile segments of the Y 2 receptor by acquiring static 15 N NMR spectra as a function of CP contact time as well as directly excited 15 N NMR spectra24. It could be shown that static 15 N CP spectra recorded at a CP contact time of 8 ms can be readily quantified to provide the relative proportion of relatively rigid and highly mobile residues24.…”

“…This investigation came to the conclusion that the receptor undergoes axially symmetric rotational diffusion in the membrane and about 10% of the residues are subject to larger amplitude motions. In contrast, the molecular dynamics of the human neuropeptide Y receptor type 2 has been systematically studied revealing that the individual segments of the molecule are subject to large amplitude motions, expressed by average order parameters between 0.55 and 0.67 in the protein backbone, which corresponds to amplitudes of the segmental motions up to about 50° 2324. Here, we report results on the molecular dynamics of the human growth hormone secretagogue (GHS) receptor, which plays a key role in the regulation of appetite and food intake and, therefore, represents an interesting target for intervening with eating disorders25.…”

Studying dynamics by magic-angle spinning solid-state NMR spectroscopy: Principles and applications to biomolecules

“…There is a growing number of interesting dynamics studies that address proteins of increasing complexity and biological interest, such as membrane proteins [104,184,185] and amyloid fibrils [106,186,187]. For reasons of limited space we focus here only on one particular aspect of biomolecular dynamics.…”

Juxta‐terminal Helix Unwinding as a Stabilizing Factor to Modulate the Dynamics of Transmembrane Helices

“…As opposed to being passive connectors, the loops and other inter-helix segments could assume added importance for establishing the conformations and regulating the functions of prominent classes of membrane proteins such as, for example, the seven-helix G-protein-coupled receptors [31–32] . It has been observed, for example, that helix fraying is a mechanism by which multi-span proteins such as G-protein-coupled receptors may adjust to a decrease in the hydrophobic thickness of the surrounding membrane [33–34] .…”