Although the oligomeric states of G-protein-coupled receptors (GPCRs) and interactions with cognate G proteins are central to their signal transduction capabilities, they remain poorly defined. In this study, we used small-angle neutron scattering (SANS) and a neutron contrast matching approach to elucidate the oligomeric states of the archetypal GPCR, rhodopsin, and its interaction with the G protein transducin (Gt). At a rhodopsin/lipid molar ratio of 1/360, we found that dark-adapted rhodopsin exists as a monomer, a finding consistent with its high functional activity measured upon photoactivation by spectrophotometry and the rate of catalyzed [35S]-GTP-γ-S exchange. Following light activation, we observed that rhodopsin forms a stable 1:1 stoichiometric complex with Gt, the structure of which is consistent with recent cryo-EM data. In contrast, activated rhodopsin in the absence of Gt showed a propensity to form higher order oligomers. This research underscores the concentration-dependent nature of rhodopsin oligomerization and establishes SANS and the ability to produce appropriately contrast-matched samples, as a robust strategy for characterizing integral membrane protein interactions under biologically relevant conditions.

Two-photon polarization ratio ()Ω measures the angle between optical transition dipole moment and permanent dipole moments difference. Linking two VenusA206 copies together results in changes of Ω value, reflecting different dipoles arrangement in excitonically coupled chromophores.

Tremendous progress has been made in determining the structures of G-protein coupled receptors (GPCR) and their complexes in recent years. However, understanding activation and signaling in GPCRs is still challenging due to the role of protein dynamics in these processes. Here, we show how dynamic nuclear polarization (DNP)-enhanced magic angle spinning nuclear magnetic resonance in combination with a unique pair labeling approach can be used to study the conformational ensemble at specific sites of the cannabinoid receptor 2. To improve the signal-to-noise, we carefully optimized the DNP sample conditions and utilized the recently introduced AsymPol-POK as a polarizing agent. We could show qualitatively that the conformational space available to the protein backbone is different in different parts of the receptor and that a site in TM7 is sensitive to the nature of the ligand, whereas a site in ICL3 always showed large conformational freedom.

Under physiological conditions fluorescent proteins (FP) in close proximity unexpectedly exhibit photophysical effects that are consistent with excitonic coupling: (1) ultrafast drop in anisotropy, (2) Davydov splitting in its CD spectra and (3) multiple FPs behaving as a single quantum emitter in photon antibunching experiments. We hypothesize that the FP β-barrel structure protects its internal chromophore from environmental decoherence, and thus slows its dephasing time relative to its energy transfer rate.

We monitored the effect on function of the G-protein-coupled receptor (GPCR) rhodopsin from small, stepwise changes in bilayer thickness induced by cholesterol. Over a range of phosphatidylcholine bilayers with hydrophobic thickness from ≈21 Å to 38 Å, the metarhodopsin-I (MI)/metarhodopsin-II (MII) equilibrium was monitored with UV-visible spectroscopy while ordering of hydrocarbon chains was probed by 2H-NMR. Addition of cholesterol shifted equilibrium toward MII for bilayers thinner than the average length of hydrophobic transmembrane helices (27 Å) and to MI for thicker bilayers, while small bilayer thickness changes within the range of the protein hydrophobic thickness drastically up- or downregulated MII formation. The cholesterol-induced shifts toward MII for thinner membranes correlated with the cholesterol-induced increase of bilayer hydrophobic thickness measured by NMR, consistent with continuum elastic modeling. The energetic penalty of adding cholesterol to thick bilayers caused rhodopsin oligomerization and a shift toward MI. In membranes of physiological thickness, changes in bilayer mechanical properties induced by cholesterol potentiated the interplay between bilayer and protein thickness resulting in large swings of the MI-MII equilibrium. In membrane containing cholesterol, elastic deformations near the protein are a dominant energetic contribution to the functional equilibrium of the model GPCR rhodopsin.

Cannabinoid receptors belong to the class of G protein-coupled receptors (GPCR) that regulate a multitude of physiological processes by transmitting signals from the extracellular milieu to intracellular proteins. They are primary targets of pharmaceutical drug development. Spectroscopic studies like EPR are ideally suited to follow the conformational dynamics of the receptor and changes upon ligand binding. We labeled specific cystein residues of the receptor with 3-(2-Iodoacetamido)-PROXYL spin label by replacing naturally occurring reactive cysteines and introducing a new single cysteine at selected sites.

Synaptic functions are mediated and modulated by a coordinated choreography of protein conformational changes and interactions in response to intracellular calcium dynamics. Time-lapse Förster resonance energy transfer can be used to study the dynamics of both conformational changes and protein-protein interactions simultaneously under physiological conditions if two resonance energy transfer reactions can be multiplexed. Binary-FRET is a technique developed to independently monitor the dynamics of calcium-calmodulin dependent protein kinase-II catalytic-domain pair separation in the holoenzyme, and its role in establishing activity-dependent holoenzyme affinity for the NR2B binding fragment of the N-methyl-D-aspartate receptor. Here we show that a transient excited-state intermediate exists where paired catalytic-domains in the holoenzyme first separate prior to subsequent NR2B association. Additionally, at non-saturating free calcium concentrations, our multiplexed approach reveals that the holoenzyme exhibits a biochemical form of plasticity, calcium dependent adaptation of T-site ligand binding affinity.

Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB2 by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB2 in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB2.

Rational design of pharmaceutical drugs targeting integral membrane G protein-coupled receptors (GPCR) requires thorough understanding of ligand binding and mechanism of activation through high resolution structural studies of purified proteins. Due to inherent conformational flexibility of GPCR, stabilization of these proteins solubilized from cell membranes into detergents is a challenging task. Here, we take advantage of naturally occurring post-translational modifications for stabilization of purified GPCR in detergent micelles. The recombinant cannabinoid CB2 receptor was expressed at high yield in Expi293F mammalian cell cultures, solubilized and purified in Façade detergent. We report superior stability of the mammalian cell-expressed receptor compared to its E. coli-expressed counterpart, due to contributions from glycosylation of the N terminus and palmitoylation of the C terminus of CB2. Finally, we demonstrate that the mammalian Expi293F amino acid labelling kit is suitable for preparation of multi-milligram quantities of high quality, selectively stable isotope-labeled GPCR for studies by nuclear magnetic resonance.

G protein?coupled receptors (GPCRs) comprise a large class of integral membrane proteins involved in the regulation of a broad spectrum of physiological processes and are a major target for pharmaceutical drug development. Structural studies can help advance the rational design of novel specific pharmaceuticals that target GPCRs, but such studies require expression of significant quantities of these proteins in pure, homogenous, and sufficiently stable form. An essential precursor for these structural studies is an assessment of protein stability under experimental conditions. Here we report that solubilization of a GPCR, type II cannabinoid receptor CB2, in a Fa?ade detergent enables radioligand thermostability assessments of this receptor with low background from nonspecific interactions with lipophilic cannabinoid ligand. Furthermore, this detergent is compatible with a [S-35]GTP?S radionucleotide exchange assay measuring guanine exchange factor activity that can be applied after heat treatment to further assess receptor thermostability. We demonstrate that both assays can be utilized to determine differences in CB2 thermostability caused by mutations, detergent composition, and the presence of stabilizing ligands. We report that a constitutively active CB2 variant has higher thermostability than the WT receptor, a result that differs from a previous thermostability assessment of the analogous CB1 mutation. We conclude that both ligand-binding and activity-based assays under optimized detergent conditions can support selection of thermostable variants of experimentally demanding GPCRs.

The human CB2 receptor belongs to the large family of rhodopsin-like G protein-coupled membrane receptors (GPCR) which are critical for transmitting external signals to the cell interior. Oligomerization of GPCR has been recognized as important for modulating the function of signaling pathways. Currently, there is a lack of effective methods that allow studying receptor oligomerization under physiologically relevant conditions, which include a fluid lipid matrix of proper composition and higher receptor concentrations. We used small angle neutron scattering (SANS) in combination with neutron scattering length density (NSLD) contrast matching of the lipid matrix into which the protein is imbedded for measurement of GPCR oligomerization. In order to obtain a SANS signal of a membrane protein of highest intensity, the difference between neutron scattering length densities (NSLD) of protein and lipid was maximized. Furthermore, the NSLD of the lipid matrix was matched to the NSLD of the buffer such that the SANS spectrum is dominated by the protein. Overall proton content of the sample was minimized to reduce incoherent scattering of neutrons that obscures the data at higher angles of diffraction. Optimal conditions were achieved by conducting experiments on protonated protein imbedded into an almost entirely perdeuterated lipid matrix in the presence of close to 100 % perdeuterated buffer. Experimental results are compared to the structure of CB2 in a lipid matrix predicted by molecular simulations.

G protein-coupled membrane receptors (GPCR) transmit extracellular signals elicited by compounds like neural transmitters, hormones, odorants, or light to the cell interior, where they activate GTP-binding proteins (G proteins). It is desirable to study GPCR and G protein under functional conditions in their natural environment, namely a fluid lipid matrix. Experiments were conducted using the GPCR bovine rhodopsin and the G protein transducin. We have recently shown that SANS is capable of measuring the state of GPCR homo-oligomerization in a lipid matrix under functional conditions. Moreover, these experiments proved that the resolution is sufficient to obtain information about the size and shape of membrane associated protein-protein complexes. We conducted detailed, time-resolved functional studies of G protein activation by GPCR via measurement of GDP to radioactive [S35] GTP-γ-S exchange. The experiments revealed experimental conditions at which homogeneity and stability of the G protein-GPCR complex are sufficient for conducting experiments on the timescale of several hours as required for small angle neutron scattering (SANS) and at a protein/lipid molar ratio that is expected yielding lowest perturbation of SANS from the presence of a lipid matrix. The following questions are addressed: Does the water-soluble G protein bind directly to GPCR in liposomes from solution or does it bind to the lipid-water interface before interacting with GPCR? Is GPCR-G protein interaction sensitive to the conformational state of GPCR switched by ligands? Does G protein interact with individual GPCR molecules or with GPCR dimers? Interaction of G protein with agonist bound GPCR triggers a GDP-to-GTP exchange at the Gα subunit of the GPCR and a dissociation of Gα and Gβγ subunits. What is the affinity of Gα and Gβγ subunits for each other, for interaction with the lipid matrix and the GPCR?

Human cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) is implicated in an array of health related systemic body functions including the immune response, inflammation, and pain sensing, and it is an important target for pharmaceutical drug development. Structural and functional studies of GPCR by NMR, EPR and fluorescence techniques require non-intrusive, site-specific labeling of the protein. This can be achieved by chemical labeling of cysteines of the receptor. In this study we explored feasibility of labeling of selected cysteines by minimizing the number of reactive SH groups through systematic replacement of the majority of the 13 naturally-occurring cysteines of CB2. The replacement of the six water-exposed cysteines resulted in a well-expressed, fully functional receptor as evidenced by ligand binding- and G protein activation studies. While the replacement of four additional cysteines in the transmembrane domain of the protein lowered ligand binding affinity several fold, the receptor could still be activated by an excess of a high affinity agonists and performed competently in an in vitro G protein activation assay. The reduced cysteine templates of CB2 were either labeled directly or used to introduce new cysteines in selected positions at the extracellular and intracellular surfaces of the protein. The cysteine mutants of CB2 were chemically labeled with 2,2,2-trifluoroethanethiol (TET) and their spectra recorded by 19F NMR in micelles composed of dodecyl maltoside and CHAPS or in particles formed by the NVoy polymer. The latter was advantageous since it yielded monomeric receptor suitable for recording solution-state 19F NMR spectra with decent resolution. This study is a precursor for exploring the structure-function relationship of CB2 by NMR and EPR.

G Protein Coupled Receptor (GPCR) dimerization has emerged as an essential mechanism regulating GPCR biosynthesis, maturation, ligand binding, coupling with G protein and downstream signaling in many cell-signaling pathways. However, determining the oligomeric state of a GPCR in a membrane is in itself a challenge, due to the complex nature of the plasma membrane and spontaneous receptor collisions. The protonated G protein-coupled membrane receptor bovine rhodopsin was reconstituted into proteoliposomes composed of perdeuterated lipids extracted from E. coli bacteria grown on fully deuterated medium. It was observed that intensity of small-angle neutron scattering (SANS) from entire proteoliposomes in 98% D2O buffer, measured at angles q<0.008 Å−1 reached a minimum at a protein/deuterated lipid molar ratio near 1/500, while yielding maximal scattering contrast for incorporated opsin (bleached rhodopsin) over the wider angular range of 0.008 Å−1

It was established previously that the G protein-coupled membrane receptor rhodopsin has transmembrane helices which match a hydrophobic bilayer thickness of 27±1 Å. Here we demonstrate that small changes of bilayer thickness of ±2 Å about that match point translate in the considerable changes of rhodopsin activation measured as the metarhodopsin I (MI)/metarhodopsin II (MII) equilibrium. We observed a biphasic behavior of the MI/MII equilibrium, with a sharp decline towards MI from 25-27 Å followed by a rapid increase of MII from 27-29 Å. Results are qualitatively identical for thickness changes induced by mixing of 16:0-16:1 PC and 18:0-18:1 PC, or 16:1-16:1 PC and 18:1-18:1 PC, or addition of 0-30 mol% cholesterol to 16:0-16:1 PC. The biphasic behavior was observed regardless of lipids used to alter bilayer hydrophobic thickness suggesting a relationship between small changes in hydrophobic thickness and rhodopsin function. It strongly favors an explanation based on a change of elastic stresses in lipid bilayers upon the transition from negative curvature in lipid monolayers near the protein below 27 Å hydrophobic thickness to positive monolayer curvature above the match point. A continuum elastic model of the membrane, including the effect of lipid monolayer curvature near the protein, predicts membrane mediated clustering of rhodopsin and stabilization of the MI photointermediate at the matching point. Small, physiologically relevant changes in cholesterol content of bilayers with a thickness in the physiologically relevant range do drastically down- or up regulate the amount of MII which is the state that activates G protein.

Lipid composition of the membrane and rhodopsin packing density strongly modulate the early steps of the visual response of photoreceptor membranes. In this study, lipid-order and bovine rhodopsin function in proteoliposomes composed of the sn-1 chain perdeuterated lipids 14:0d27-14:1-PC, 16:0d31-16:1-PC, 18:0d35-18:1-PC, or 20:0d39-20:1-PC at rhodopsin/lipid molar ratios from 1:70 to 1:1000 (mol/mol) were investigated. Clear evidence for matching of hydrophobic regions on rhodopsin transmembrane helices and hydrophobic thickness of lipid bilayers was observed from 2H nuclear magnetic resonance order parameter measurements at low rhodopsin concentrations. Thin bilayers stretched to match the length of transmembrane helices observed as increase of sn-1 chain order, while thicker bilayers were compressed near the protein. A quantitative analysis of lipid-order parameter changes suggested that the protein adjusts its conformation to bilayer hydrophobic thickness as well, which confirmed our earlier circular-dichroism measurements. Changes in lipid order parameters upon rhodopsin incorporation vanished for bilayers with a hydrophobic thickness of 27 ± 1 Å, suggesting that this is the bilayer thickness at which rhodopsin packs in bilayers at the lowest membrane perturbation. The lipid-order parameter studies also indicated that a hydrophobic mismatch between rhodopsin and lipids triggers rhodopsin oligomerization with increasing rhodopsin concentrations. Both hydrophobic mismatch and rhodopsin oligomerization result in substantial shifts of the equilibrium between the photointermediates metarhodopsin I and metarhodopsin II; increasing bilayer thickness favors formation of metarhodopsin II while oligomerization favors metarhodopsin I. The results highlight the importance of hydrophobic matching for rhodopsin structure, oligomerization, and function.