Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2026 Jun 27.
Published in final edited form as: Curr Opin Struct Biol. 2025 Jun 27;94:103103. doi: 10.1016/j.sbi.2025.103103

The influence of lipids and biological membranes on the conformational equilibria of GPCRs: insights from NMR spectroscopy

Greeshma Jain 1, Matthew T Eddy 1
PMCID: PMC12264821  NIHMSID: NIHMS2094288  PMID: 40580801

Abstract

G protein-coupled receptors (GPCRs) function within cellular membranes, complex and dynamic environments. Rather than serving as a passive background, lipid membranes actively influence GPCR drug responses and signaling. Studies utilizing nuclear magnetic resonance (NMR) spectroscopy have revealed key insights into receptor-lipid interactions, enabled by the compatibility of NMR experiments with many different membrane systems and physiological temperature, conditions more closely reflecting the native cellular environment. NMR data have revealed new mechanistic insights that explain how specific lipids regulate GPCR activation, how bulk membrane properties influence receptor dynamics, and how different membrane mimetics affect GPCR behavior. These findings establish a framework for bridging in vitro structural studies with in vivo biological and pharmacological data.

Introduction

G protein-coupled receptors (GPCRs) are crucial sensory integral membrane proteins. Earlier structural and biophysical studies focused on elucidating mechanisms of receptor–ligand and receptor–protein molecular recognition, specifically how extracellular ligands and intracellular signaling partners engaged the receptor [1-3]. More recently, attention has shifted toward the integral role of the membrane environment itself, particularly how lipid-receptor interactions modulate ligand binding and agonist-induced signaling [4]. A growing body of structural and functional evidence supports the view that lipids can profoundly influence GPCR activity, not only as allosteric or orthosteric modulators, but also through bulk membrane properties such as curvature [5] and fluidity. Insights into GPCR-lipid interactions provide a critical framework for bridging in vitro structural and pharmacological observations with the receptor's physiological functions in vivo.

NMR spectroscopy has a long-standing history studying biological membranes and lipid–protein interactions, with pioneering investigations of lipid bilayers dating back over five decades [6-8]. More recently, NMR has emerged as a powerful tool for elucidating the mechanisms of G protein-coupled receptor (GPCR) signaling by enabling the observation of multiple conformational states in equilibrium and revealing how small-molecule ligands modulate these conformational landscapes, as has been reviewed [9-13]. Important advantages of NMR include the flexibility to study membrane proteins in a wide range of membrane or membrane-mimetic systems and at physiologically relevant temperature. GPCR studies have increasingly leveraged these strengths to uncover new mechanistic insights into how lipids and biological membranes influence receptor conformational equilibria.

This review highlights recent contributions from NMR spectroscopy to our current understanding of how biological membranes and lipids influence GPCR signaling. We discuss NMR studies that directly probe GPCRs as well as investigations into the effects of membrane environments on GPCR interactions with their partner signaling proteins. Examples from the literature illustrate the application of diverse NMR methodologies, including 19F-NMR and multi-dimensional experiments enabled by various stable-isotope labeling strategies. Insights derived from both solid-state and solution NMR approaches are discussed, with an emphasis on unique strengths of each method. Collectively, these studies underscore how NMR has advanced our molecular understanding of GPCR function by revealing how specific lipid species and bulk membrane properties influence receptor activation, modulate interactions with effector proteins, and affect the interpretation of GPCR activity in membrane mimetic systems.

Defining mechanisms underlying the impact of specific lipids on GPCR signaling

Data from structural and biophysical literature increasingly highlight the important roles of specific lipids, including tissue-specific lipids and lipids with distinct chemical properties, as modulators of GPCR activity [14,15]. A compelling example involves the fatty acids docosahexaenoic acid (DHA) and arachidonic acid (ARA), which modulate the activity of the human A2A adenosine receptor (A2AAR), a class A GPCR that regulates dopamine release. DHA and ARA together constitute ~14% of the total lipid content in the mammalian brain striatum, a region where A2AAR is extensively expressed [16]. Shimada and coworkers reported distinct changes in 2D heteronuclear correlation spectra of [[α,β,β-2H,methyl-13C] Met,u-2H] A2AAR reconstituted in lipid nanodiscs with and without DHA [17]. Spectral changes were most pronounced near the receptor intracellular surface, particularly at labeled sites in transmembrane (TM) helices 3 and 6. Based on these and additional NMR data from the same study, the authors proposed that DHA shifts the A2AAR conformational equilibrium to populate active conformational states promoting signaling complex formation, offering a mechanistic rationale for DHA-enhanced A2AAR activity [17].

In a separate study utilizing 19F-NMR spectroscopy of A2AAR in lipid nanodiscs, Eddy and colleagues investigated the mechanisms by which anionic lipids influence the activity of human A2AAR (Figure 1) [18]. One key finding was the shift in the conformational ensemble of agonist-bound A2AAR to favor a larger population of an active state in lipid nanodiscs containing a binary mixture of zwitterionic and anionic lipids. In contrast, A2AAR in lipid nanodiscs without anionic lipids showed significantly reduced population of the active conformation, consistent with a decrease in nucleotide exchange in the same conditions [18]. By integrating NMR spectroscopy, functional assays, and computational modeling and simulations, the authors proposed a mechanism in which anionic phospholipids interact with a cluster of charged amino acids near the intracellular surface of the receptor [18]. This cluster also engages with the C-terminal region of G proteins, thereby promoting G protein recognition and the formation of ternary signaling complexes. These charged residues, located at positions 6.32 and 6.35 (numbers refer to the Ballesteros-Weinstein nomenclature [19]) were found to be highly conserved not only among class A GPCRs, but across all GPCR classes (Figure 1), suggesting a potentially general mechanism for receptor-lipid interactions that regulate signaling activity [18].

Figure 1.

Figure 1

Open in a new tab

Insights from NMR into the impact of anionic lipids on the conformational equilibria of the human A2A adenosine receptor (A2AAR). (a) The 1D 19F-NMR spectra of agonist-bound human A2AAR in lipid nanodiscs containing different defined mixtures of lipids. The yellow peak labeled P4 is an active conformational state observed in the presence of anionic lipids but missing in spectra of the agonist-bound receptor in nanodiscs containing only zwitterionic lipids. (b) 19F-NMR spectra of agonist-bound A2AAR in nanodiscs containing POPC. The active conformational state P4 is observed upon addition of an engineered GαS protein, however the population of this state is lower than when in the presence of anionic lipids. (c) Schematic of the impact of anionic lipids on the conformational equilibria of apo, antagonist-bound, and agonist-bound A2AAR, showing that anionic lipids populate an active-like conformation of the receptor that recognizes the G protein. (d) Conservation of residues at the intracellular end of TM VI among 290 class A GPCRs. The size of each amino acid corresponds to its frequency of occurrence at that position. Numbers indicate the residue positions in the Ballesteros-Weinstein nomenclature. The figures in all panels were adapted from reference 18, with permission.

Ziarek and co-workers used 13CεH3-methionine NMR to investigate the impact of the anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2), a key modulator of GPCR signaling, on the conformational dynamics of the neurotensin receptor 1 (NTS1) [20]. They found that PIP2 interactions with the intracellular surface of NTS1 allosterically modulated conformational dynamics at the receptor orthosteric binding pocket and conserved activation hot spots [20]. A subsequent NMR study of NTSR1 showed that zwitterionic lipids stabilized the secondary structure of the amphipathic helix 8 but detergents did not [21].

Huster and colleagues have conducted solid-state NMR studies of the neuropeptide Y2 receptor (NPY2R), including comparative analyses of receptor conformational dynamics in membrane environments composed of saturated DMPC lipids [22] and monounsaturated POPC lipids [23]. A key finding was that NPY2R exhibited greater rigidity when reconstituted in POPC membranes compared to DMPC membranes, suggesting increased receptor order correlated with either longer lipid acyl chains or lipid saturation and may be associated with enhanced receptor helical content [23].

Mechanistic insights into cholesterol-mediated regulation of GPCR signaling

Because of its well-documented role in regulating membrane fluidity and its frequent association with GPCRs in crystal and cryo-EM structures, cholesterol has been a special focus in lipid-receptor NMR studies. Cholesterol–receptor interactions are complex, with cholesterol and its analogs thought to act as either agonists or antagonists. These multifaceted roles have also been investigated through computational simulations [24-27]. A key unresolved question remains whether cholesterol influences GPCR function through direct receptor-cholesterol interactions or indirectly by altering the bulk physical properties of the membrane. Utilizing 19F-NMR spectroscopy with the human A2AAR, Prosser and co-workers observed that cholesterol behaved as a weak allosteric modulator, enhancing A2AAR signaling activity [28] (Figure 2a). These findings were further supported by 19F-NMR experiments recorded with increasing applied pressure, which mimicked the ordering effects of cholesterol on the lipid bilayer. With increasing pressure, 19F-NMR data showed a shift in the A2AAR conformational ensemble that favored an increased population of an active state [28], consistent with observations that increased membrane order enhanced A2AAR nucleotide exchange [28].

Figure 2.

Figure 2

Open in a new tab

Changes in lipid bilayer properties, driven by the presence of cholesterol or applied external pressure, manifested as NMR-observed changes in GPCR conformational equilibria. (a) 19F-NMR observations of the impact of applied external pressure, which is used to modulate bulk properties of lipid membranes. One-dimensional 19F-NMR spectra of A2AAR, labeled at position V229C, in lipid nanodiscs recorded over a range of increasing applied pressures. Functional states are annotated and indicated by the grey dashed lines. Increasing pressure increased the population of active conformational ensembles, as indicated by the increase in signal intensity for the state A1. (b) NMR observations of the impact of pressure and CHS on the conformation and structural plasticity of β1AR, as observed in superimposed 2D [15N,1H]-TROSY spectra. Resonance assignments are annotated on the spectra. The dashed lines connect residues with two resonances observed for a preactive state, ‘p’, and active conformational state, ‘a’. Panels a and b adapted from references 24 and 27, respectively, with permission.

In a subsequent 19F-NMR study of A2AAR in lipid nanodiscs, Eddy and colleagues proposed a model of cholesterol’s influence on A2AAR activity through both indirect influence on membrane properties and direct receptor-cholesterol interactions [29]. In the absence of anionic lipids, cholesterol and cholesterol analogs significantly increased the population of the A2AAR active conformational state. Cholesterol-driven activation occurred even for nanodiscs containing only 1-2 molecules of cholesterol per nanodisc, supporting the idea that specific receptor-cholesterol interactions influenced receptor activation [29]. However, in the presence of anionic lipids, the impact of cholesterol on A2AAR activation was marginal, suggesting that in these conditions, cholesterol’s impact may be more indirect [29], aligning with similar conclusions by Prosser and colleagues [28]. In a separate study of the β2-adrenergic receptor (β2AR), saturation-transfer NMR experiments by Milon and colleagues indicated that β2AR interacted with cholesterol but not with the structurally similar molecule ergosterol [30], suggesting that GPCR-cholesterol interactions were specific for β2AR.

The influence of the cholesterol analog cholesterol hemisuccinate (CHS), which is frequently used in GPCR structural and biophysical studies, was investigated in NMR experiments with 15N-Val labeled β1-adrenergic receptor (β1AR) [31] (Figure 2b). Two-dimensional TROSY NMR spectra of β1AR showed that without CHS, increasing pressure shifted the β1AR conformational equilibrium toward an active conformation. Addition of CHS blocked pressure-induced β1AR activation, supporting the idea that CHS acted as a negative allosteric modulator of β1AR signaling. This result suggested that even among class A receptors, different GPCRs may be influenced by cholesterol and cholesterol analogs in surprisingly different ways.

The influence of cholesterol on the chemokine receptor CCR3 has also been explored. Functional studies by Wylie and colleagues demonstrated that increasing cholesterol concentrations in SMALPs and lipid vesicles enhanced both GTP hydrolysis and binding of the chemokine CCL11 [32]. Follow-up modeling and multi-dimensional NMR studies using uniformly 15N,13C-labeled CCR3 revealed cholesterol-induced alterations in the conformation and dynamics of extracellular loop 2 (ECL2), ECL3, and the transmembrane helices [33], offering mechanistic insights into how cholesterol modulates CCR3 function through conformational changes.

Elucidating the effects of lipid membranes on GPCR recognition and interactions with partner signaling proteins

Evidence from structural, biophysical, and functional studies [34-37] support the idea that lipid membranes play an active role facilitating and controlling interactions of receptors with partner signaling proteins, especially with G proteins and β-arrestins. Using 19F-NMR spectroscopy, Prosser and co-workers investigated conformational changes with the stimulatory G protein α subunit, GαS, upon interacting with phospholipid membranes and A2AAR (Figure 3a and 3b) [38]. One of the key results from this work showed that the N-terminus of GαS interacted with the lipid bilayer even in the absence of a palmitoylated GαS N-terminus, driving conformational changes at additional locations in GαS that facilitated complex formation with A2AAR [38].

Figure 3.

Figure 3

Open in a new tab

The influence of membrane lipids on GPCR interactions with partner proteins as viewed by 19F-NMR. (a) Crystal structure of the trimeric GS protein in complex with GDP. GαS is shown in ribbon representation with several sites selected for 19F-NMR labeling shown as colored spheres and annotated with the residue numbers. Gβ and Gγ are shown in pink and blue surface representations, respectively. (b) The conformational landscape of GαS viewed by 19F-NMR for GαS labeled at several distinct single cysteine positions. Experimental conditions included GαS in the presence of lipid nanodiscs, denoted as “membrane”, and lipid nanodiscs containing A2AAR. Horizontal lines indicate two global conformational states observed in all spectra. (c) NMR spectra of 19F-labeled at position H295C in the gate loop of βarr1. Spectra were recorded in different conditions, including in the presence of the lipid PIP2. (d) NMR spectral comparison of several 19F-labeled sites of βarr1 in the presence and absence of lipid nanodiscs, labeled as “Membrane”. (e) 19F-NMR spectra showing the impact of PIP2 interactions with βarr1. (f) Crystal structure of βarr1 with positions viewed by 19F-NMR shown as circles, which are colored according to the extent of perturbation to the NMR signals due to the presence of lipid nanodiscs. Panels a and b adapted from reference 34, and c–f adapted from reference 35, with permission.

NMR studies have also investigated the role of phospholipid membranes on the interactions between receptors and β-arrestins and especially arrestin-2, also known as βarr1. A 19F-NMR study by Hu and coworkers observed distinct changes in the conformation of βarr1 upon interaction with PIP2 and the formation of an intermediate conformational state, which was proposed to be an activation mechanism distinct from interactions with the phosphorylated receptor C-terminus (Figure 3c-f) [39]. Shimada and coworkers utilized 2D HMQC NMR to observe deuterated and selectively isoleucine-methylated βarr1 in the presence of lipid nanodiscs and a chimeric version of the β2AR, finding that changes in NMR spectra were not observed for arrestin in the presence of lipids in nanodiscs without receptor, but that lipids were necessary for facilitating complex formation with the chimeric receptor [40].

Delineating the influence of membrane mimetic systems on GPCR structure and conformational dynamics

The practical challenges associated with purifying membrane proteins for structural and biophysical studies have driven the development of a variety of membrane and membrane-mimetic systems, including detergents, nanodiscs, and lipid vesicles. These systems aim to provide a stable environment that preserves the native structure and function of membrane proteins, but it is increasingly appreciated that these systems can unintentionally impact the structures and conformational dynamics of membrane proteins. An earlier study by Prosser and coworkers compared the conformational equilibria of β2AR between two different detergent systems, concluding that the choice of detergent exhibited a significant influence on the rates of exchange among different β2AR conformational states in 19F-NMR spectra [41]. Shimada and colleagues compared the conformational equilibria of β2AR between in DDM micelles versus lipid nanodiscs (Figure 4a) [42]. In two-dimensional [13C,1H]-HMQC spectra of selectively deuterated and methyl-13C-Met labeled β2AR, they observed changes in the chemical shifts and relative intensities of methionine signals between detergent and nanodisc preparations for complexes of β2AR with different ligands. These differences reflected variations in the rates of exchange among conformational states that depended on the surrounding environment (Figure 4a) [42]. Lefkowitz, Prosser and colleagues observed differences in the conformational equilibria of β2AR in 19F-NMR spectra comparing detergent and lipid nanodisc preparations [43], which were attributed to differences in the constitutive activity levels of the receptor between different environments.

Figure 4.

Figure 4

Open in a new tab

NMR comparison of the impact of different membrane mimetic environments on GPCR conformation and structural plasticity. (a) Comparison of 2D [13C,1H]-HMQC spectra of selectively deuterated and methyl-13C-Met β2AR complexes with ligands of different efficacies in reconstituted high-density lipoproteins (rHDL), i.e., lipid nanodiscs, and DDM detergent micelles. NMR resonances from M82 of different β2AR–ligand complexes are superimposed. (b) Schematic comparing the β2AR conformational states and rates of conformational exchange observed by NMR between rHDL and detergent micelle preparations. Conformations with populations less than 10% are shown in fainter colors. (c) Two-dimensional [15N,1H]-TROSY spectra of an A2AAR complex with an antagonist in LMNG detergent micelles (red) and lipid nanodiscs (blue). (d) Superimposed expanded 2D sections from the dashed boxes shown in (c). Assigned tryptophan residues are annotated. (e) One-dimensional cross sections taken from the plots shown in (d) comparing the chemical shifts and line widths of highlighted tryptophan residues between detergent and lipid nanodisc preparations. Panels a and b adapted from reference 38, and panels c–e adapted from reference 40, with permission.

The conformation of antagonist-bound A2AAR was compared between LMNG/CHS and lipid nanodisc preparations in a study by the Wüthrich group (Figure 4b) [44]. Two-dimensional TROSY spectra of [u-15N, ~70% 2H]-A2AAR in complex with ZM241385 were similar between LMNG/CHS and nanodisc preparations, suggesting that the receptor adopted comparable conformations in these two environments (Figure 4b). The Wüthrich group further assessed the conformational equilibria of A2AAR between detergent and lipid nanodisc preparations using 19F-NMR, concluding that the conformational ensemble of A2AAR measured at 25 °C in lipid nanodiscs more closely resembled that observed in LMNG/CHS than in DDM/CHS micelles [45].

Eddy and colleagues used 19F-NMR to compare the conformational equilibria of A2AAR between detergent micelle and lipid nanodisc preparations, including a systematic comparison of the conformational equilibria measured over a range of temperatures [46]. An important result from this study was that the active state population of A2AAR observed in 19F-NMR spectra quantitatively correlated with measured cAMP data across a range of ligands with different efficacies for A2AAR in lipid nanodiscs measured at physiological temperature but not for A2AAR in DDM/CHS detergent. In a subsequent study, Eddy and coworkers used magic angle spinning (MAS) solid-state NMR to observe the conformational equilibria of A2AAR in lipid vesicles of defined compositions[47]. Comparison of the conformational equilibria of A2AAR between lipid nanodiscs, detergent micelles, and vesicles showed similar conformational equilibria for antagonist-bound A2AAR but different relative populations for the agonist-bound conformational ensemble in the different membrane systems [47], suggesting that different bulk membrane properties may impact GPCR conformational equilibria.

Conclusions and Outlook

NMR studies have provided compelling evidence that lipids and lipid membranes significantly influence the conformational equilibria of GPCRs and their interactions with partner proteins. To date, NMR studies of GPCR-lipid interactions have mostly focused on class A receptors, and exciting opportunities will arise by studying receptors from additional classes. NMR experiments are capable of more precisely characterizing lipid-interacting sites on receptors and quantitatively measuring the lifetimes of lipid-receptor interactions, following related work with channel proteins [48-50]. Future studies leveraging these capabilities could provide insights that support the intriguing possibility of targeting lipid-interacting sites with small molecule allosteric modulators.

Improved understanding of how lipids and lipid membranes affect receptor signaling will likely drive improvement of pharmacological applications, for example by clarifying how cellular membrane environments influence the measurement of drug efficacy or enabling identification of novel small molecules. A recent application of high-resolution magic angle spinning (HRMAS) NMR with A2AAR in unpurified cell membranes demonstrated that native cell membrane environments can improve sample stability and small molecule ligand screening in more physiologically relevant conditions [51]. The NMR studies of lipid-receptor interactions reviewed here lay the foundation for the long-term goal of investigating GPCR structure and dynamics directly within the native cellular environment. Achieving this goal will likely benefit from breakthroughs that improve signal-to-noise in NMR experiments, including through dynamic nuclear polarization (DNP) NMR, which has been utilized to demonstrate the feasibility of probing structure and dynamics of bacterial membrane proteins within their native contexts [52-54].

Acknowledgements

This work was supported by the National Institutes of Health grant R35GM138291.

Footnotes

Conflict of interest statement

None declared

References

  • 1.Pandey A, Larda ST, La Plante S, Prosser RS: NMR-based approaches to the study of GPCRs and GPCR-ligand interactions. In GPCRs. Edited by: Elsevier; 2020:65–80. [Google Scholar]
  • 2.Conflitti P, Lyman E, Sansom MSP, Hildebrand PW, Gutiérrez-de-Terán H, Carloni P, Ansell TB, Yuan S, Barth P, Robinson AS, et al. : Functional dynamics of G protein-coupled receptors reveal new routes for drug discovery. Nature Reviews Drug Discovery 2025, 24:251–275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Ueda T, Kofuku Y, Okude J, Imai S, Shiraishi Y, Shimada I: Function-related conformational dynamics of G protein–coupled receptors revealed by NMR. Biophysical Reviews 2019, 11:409–418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Jones AJY, Gabriel F, Tandale A, Nietlispach D: Structure and dynamics of GPCRs in lipid membranes: physical principles and experimental approaches. Molecules 2020, 25:4729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Rosholm KR, Leijnse N, Mantsiou A, Tkach V, Pedersen SL, Wirth VF, Oddershede LB, Jensen KJ, Martinez KL, Hatzakis NS, et al. : Membrane curvature regulates ligand-specific membrane sorting of GPCRs in living cells. Nature Chemical Biology 2017, 13:724–729. [DOI] [PubMed] [Google Scholar]
  • 6.Oldfield E, Chapman D: Dynamics of lipids in membranes: heterogeneity and the role of cholesterol. FEBS letters 1972, 23:285–297. [DOI] [PubMed] [Google Scholar]
  • 7.Griffin RG: Solid state nuclear magnetic resonance of lipid bilayers. In Methods in Enzymology. Edited by: Elsevier; 1981:108–174. vol 72.] [DOI] [PubMed] [Google Scholar]
  • 8.Brown MF, Ribeiro AA, Williams GD: New view of lipid bilayer dynamics from 2H and 13C NMR relaxation time measurements. Proceedings of the National Academy of Sciences 1983, 80:4325–4329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Silva LO, Wijesekara AV, Eddy MT: NMR spectroscopy reveals insights into mechanisms of GPCR signaling. Trends in Biochemical Sciences 2024:S0968000424002329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Jin B, Thakur N, Wijesekara AV, Eddy MT: Illuminating GPCR signaling mechanisms by NMR spectroscopy with stable-isotope labeled receptors. Current Opinion in Pharmacology 2023, 72:102364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ye L, Wang X, McFarland A, Madsen JJ: 19F NMR: A promising tool for dynamic conformational studies of G protein-coupled receptors. Structure 2022, 30:1372–1384. [DOI] [PubMed] [Google Scholar]
  • 12.Bostock MJ, Solt AS, Nietlispach D: The role of NMR spectroscopy in mapping the conformational landscape of GPCRs. Current Opinion in Structural Biology 2019, 57:145–156. [DOI] [PubMed] [Google Scholar]
  • 13.Huang SK, Prosser RS: Dynamics and mechanistic underpinnings to pharmacology of class A GPCRs: an NMR perspective. American Journal of Physiology-Cell Physiology 2022, 322:C739–C753. [DOI] [PubMed] [Google Scholar]
  • 14.Xu P, Huang S, Zhang H, Mao C, Zhou XE, Cheng X, Simon IA, Shen D-D, Yen H-Y, Robinson CV, et al. : Structural insights into the lipid and ligand regulation of serotonin receptors. Nature 2021, 592:469–473. [DOI] [PubMed] [Google Scholar]
  • 15.Agasid MT, Robinson CV: Probing membrane protein–lipid interactions. Current Opinion in Structural Biology 2021, 69:78–85. [DOI] [PubMed] [Google Scholar]
  • 16.de Lera Ruiz M, Lim Y-H, Zheng J: Adenosine A2A receptor as a drug discovery target. Journal of medicinal chemistry 2014, 57:3623–3650. [DOI] [PubMed] [Google Scholar]
  • 17.Mizumura T, Kondo K, Kurita M, Kofuku Y, Natsume M, Imai S, Shiraishi Y, Ueda T, Shimada I: Activation of adenosine A2A receptor by lipids from docosahexaenoic acid revealed by NMR. Science Advances 2020, 6:eaay8544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Thakur N, Ray A, Sharp L, Jin B, Duong A, Gopal Pour N, Obeng S, Wijesekara A, Gao Z-G, McCurdy C, et al. : Anionic phospholipids control mechanisms of GPCR-G protein recognition. Nature Communications 2023, 14:794. ** This study integrated 19F-NMR data with computational and functional experiments to provide mechanistic insights into the impact of anionic lipids on GPCR signaling
  • 19.Ballesteros JA, Weinstein H: Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. In Methods in neurosciences. Edited by: Elsevier; 1995:366–428. vol 25.] [Google Scholar]
  • 20.Bumbak F, Bower JB, Zemmer SC, Inoue A, Pons M, Paniagua JC, Yan F, Ford J, Wu H, Robson SA, et al. : Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314. Nature Communications 2023, 14:3328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Bower JB, Robson SA, Ziarek JJ: Insights on the G protein-coupled receptor helix 8 solution structure and orientation using a neurotensin receptor 1 peptide. Protein Science 2024, 33:e4976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Schmidt P, Thomas L, Müller P, Scheidt HA, Huster D: The G-Protein-Coupled Neuropeptide Y Receptor Type 2 is Highly Dynamic in Lipid Membranes as Revealed by Solid-State NMR Spectroscopy. Chemistry – A European Journal 2014, 20:4986–4992. [DOI] [PubMed] [Google Scholar]
  • 23.Thomas L, Kahr J, Schmidt P, Krug U, Scheidt HA, Huster D: The dynamics of the G protein-coupled neuropeptide Y2 receptor in monounsaturated membranes investigated by solid-state NMR spectroscopy. Journal of Biomolecular NMR 2015, 61:347–359. [DOI] [PubMed] [Google Scholar]
  • 24.Kiriakidi S, Kolocouris A, Liapakis G, Ikram S, Durdagi S, Mavromoustakos T: Effects of cholesterol on GPCR function: insights from computational and experimental studies. Direct mechanisms in cholesterol modulation of protein function 2019:89–103. [DOI] [PubMed] [Google Scholar]
  • 25.Duncan AL, Song W, Sansom MS: Lipid-dependent regulation of ion channels and G protein–coupled receptors: insights from structures and simulations. Annual review of pharmacology and toxicology 2020, 60:31–50. [DOI] [PubMed] [Google Scholar]
  • 26.Tzortzini E, Corey RA, Kolocouris A: Comparative study of receptor-, receptor state-, and membrane-dependent cholesterol binding sites in A2A and A1 adenosine receptors using coarse-grained molecular dynamics simulations. Journal of Chemical Information and Modeling 2023, 63:928–949. [DOI] [PubMed] [Google Scholar]
  • 27.McGraw C, Koretz KS, Oseid D, Lyman E, Robinson AS: Cholesterol Dependent Activity of the Adenosine A2A Receptor Is Modulated via the Cholesterol Consensus Motif. Molecules 2022, 27:3529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Huang SK, Almurad O, Pejana RJ, Morrison ZA, Pandey A, Picard L-P, Nitz M, Sljoka A, Prosser RS: Allosteric modulation of the adenosine A2A receptor by cholesterol. eLife 2022, 11:e73901. * The use of variable applied pressure and 19F-NMR was used to assess the mechanisms of allosteric modulation of the A2A adenosine receptor by cholesterol
  • 29. Ray AP, Thakur N, Pour NG, Eddy MT: Dual mechanisms of cholesterol-GPCR interactions that depend on membrane phospholipid composition. Structure 2023, 31:836–847. * 19F-NMR was used to determine two potential mechanisms of the impact of cholesterol on the A2A adenosine receptor that depended on membrane lipid composition
  • 30.Gater Deborah L, Saurel O, Iordanov I, Liu W, Cherezov V, Milon A: Two Classes of Cholesterol Binding Sites for the β2AR Revealed by Thermostability and NMR. Biophysical Journal 2014, 107:2305–2312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Abiko LA, Dias Teixeira R, Engilberge S, Grahl A, Mühlethaler T, Sharpe T, Grzesiek S: Filling of a water-free void explains the allosteric regulation of the β1-adrenergic receptor by cholesterol. Nature Chemistry 2022, 14:1133–1141. ** NMR spectroscopy and crystallography were used to determine the mechanism of negative allosteric modulation of β1AR by cholesterol hemisuccinate
  • 32.van Aalst E, Wylie BJ: Cholesterol Is a Dose-Dependent Positive Allosteric Modulator of CCR3 Ligand Affinity and G Protein Coupling. Frontiers in Molecular Biosciences 2021, 8:724603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. van Aalst EJ, McDonald CJ, Wylie BJ: Cholesterol biases the conformational landscape of the chemokine receptor CCR3: A MAS SSNMR-filtered molecular dynamics study. Journal of Chemical Information and Modeling 2023, 63:3068–3085. * Solid-state NMR was used to investigate the impact of cholesterol on a chemokine receptor structure and conformational dynamics
  • 34.Zhang M, Gui M, Wang Z-F, Gorgulla C, Yu JJ, Wu H, Sun Z-yJ, Klenk C, Merklinger L, Morstein L, et al. : Cryo-EM structure of an activated GPCR–G protein complex in lipid nanodiscs. Nature Structural & Molecular Biology 2021, 28:258–267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Janetzko J, Kise R, Barsi-Rhyne B, Siepe DH, Heydenreich FM, Kawakami K, Masureel M, Maeda S, Garcia KC, von Zastrow M, et al. : Membrane phosphoinositides regulate GPCR-β-arrestin complex assembly and dynamics. Cell 2022, 185:4560–4573.e4519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Strohman MJ, Maeda S, Hilger D, Masureel M, Du Y, Kobilka BK: Local membrane charge regulates β2 adrenergic receptor coupling to Gi3. Nature Communications 2019, 10:2234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Staus DP, Hu H, Robertson MJ, Kleinhenz ALW, Wingler LM, Capel WD, Latorraca NR, Lefkowitz RJ, Skiniotis G: Structure of the M2 muscarinic receptor–β-arrestin complex in a lipid nanodisc. Nature 2020, 579:297–302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Huang SK, Picard L-P, Rahmatullah RSM, Pandey A, Van Eps N, Sunahara RK, Ernst OP, Sljoka A, Prosser RS: Mapping the conformational landscape of the stimulatory heterotrimeric G protein. Nature Structural & Molecular Biology 2023, 30:502–511. ** Utilizing 19F-NMR, the authors provided insights into the mechanisms underlying the influence of G protein structure and dynamics by interactions with lipids and A2AAR
  • 39. Zhai R, Wang Z, Chai Z, Niu X, Li C, Jin C, Hu Y: Distinct activation mechanisms of β-arrestin-1 revealed by 19F NMR spectroscopy. Nature Communications 2023, 14:7865. ** This study used 19F-NMR approaches to investigate mechanisms of the influence of lipids and receptor C-termini on arrestin structure and conformational dynamics
  • 40.Shiraishi Y, Kofuku Y, Ueda T, Pandey S, Dwivedi-Agnihotri H, Shukla AK, Shimada I: Biphasic activation of β-arrestin 1 upon interaction with a GPCR revealed by methyl-TROSY NMR. Nature Communications 2021, 12:7158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Chung KY, Kim TH, Manglik A, Alvares R, Kobilka BK, Prosser RS: Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor. Journal of Biological Chemistry 2012, 287:36305–36311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Kofuku Y, Ueda T, Okude J, Shiraishi Y, Kondo K, Mizumura T, Suzuki S, Shimada I: Functional Dynamics of Deuterated β2-Adrenergic Receptor in Lipid Bilayers Revealed by NMR Spectroscopy. Angewandte Chemie International Edition 2014, 53:13376–13379. [DOI] [PubMed] [Google Scholar]
  • 43.Staus DP, Wingler LM, Pichugin D, Prosser RS, Lefkowitz RJ: Detergent- and phospholipid-based reconstitution systems have differential effects on constitutive activity of G-protein–coupled receptors. Journal of Biological Chemistry 2019, 294:13218–13223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Guo C, Yang L, Liu Z, Liu D, Wüthrich K: Two-Dimensional NMR Spectroscopy of the G Protein-Coupled Receptor A2AAR in Lipid Nanodiscs. Molecules 2023, 28:5419. * The authors demonstrated that the human A2AAR in lipid nanodiscs yielded TROSY-NMR spectra of similar high quality as samples prepared in detergent micelles
  • 45.Mendoza-Hoffmann F, Guo C, Song Y, Feng D, Yang L, Wüthrich K: 19F-NMR studies of the impact of different detergents and nanodiscs on the A2A adenosine receptor. Journal of Biomolecular NMR 2024, 78:31–37. [DOI] [PubMed] [Google Scholar]
  • 46. Thakur N, Ray AP, Jin B, Afsharian NP, Lyman E, Gao Z-G, Jacobson KA, Eddy MT: Membrane mimetic-dependence of GPCR energy landscapes. Structure 2024:S0969212624000376. ** The authors demonstrate that under the right conditions, 19F-NMR of A2AAR provided results that quantiatively matched ligand efficacies measured in cellular assays
  • 47.Ray AP, Jin B, Eddy MT: The Conformational Equilibria of a Human GPCR Compared between Lipid Vesicles and Aqueous Solutions by Integrative 19F-NMR. Journal of the American Chemical Society 2025, 147:17612–17625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Borcik CG, Eason IR, Yekefallah M, Amani R, Han R, Vanderloop BH, Wylie BJ: A Cholesterol Dimer Stabilizes the Inactivated State of an Inward-Rectifier Potassium Channel. Angewandte Chemie International Edition 2022, 61:e202112232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Elkins MR, Sergeyev IV, Hong M: Determining Cholesterol Binding to Membrane Proteins by Cholesterol13 C Labeling in Yeast and Dynamic Nuclear Polarization NMR. Journal of the American Chemical Society 2018, 140:15437–15449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Elkins MR, Williams JK, Gelenter MD, Dai P, Kwon B, Sergeyev IV, Pentelute BL, Hong M: Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR. Proceedings of the National Academy of Sciences 2017, 114:12946–12951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Petracco E, Ferré G, Kabelka I, Ballante F, Carlsson J, Mulry E, Ray AP, Collins J, Allais F, Eddy MT: Development of an In Situ G Protein-Coupled Receptor Fragment Molecule Screening Approach with High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance. ACS Chemical Biology 2025, 20:401–411. [DOI] [PubMed] [Google Scholar]
  • 52.Bajaj VS, Mak-Jurkauskas ML, Belenky M, Herzfeld J, Griffin RG: Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization–enhanced solid-state NMR. Proceedings of the National Academy of Sciences 2009, 106:9244–9249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Mak-Jurkauskas ML, Bajaj VS, Hornstein MK, Belenky M, Griffin RG, Herzfeld J: Energy transformations early in the bacteriorhodopsin photocycle revealed by DNP-enhanced solid-state NMR. Proceedings of the National Academy of Sciences 2008, 105:883–888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Kent JE, Ackermann BE, Debelouchina GT, Marassi FM: Dynamic Nuclear Polarization Illuminates Key Protein–Lipid Interactions in the Native Bacterial Cell Envelope. Biochemistry 2023, 62:2252–2256. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES