A rapid, tag-free way to purify functional GPCRs.

G protein-coupled receptors (GPCRs) play diverse signaling roles and represent major pharmaceutical targets. Consequently, they are the focus of intense study, and numerous advances have been made in their handling and analysis. However, a universal way to purify GPCRs has remained elusive, in part because of their inherent instability when isolated from cells. To address this, we have developed a general, rapid, and tag-free way to purify GPCRs. The method uses short peptide analogs of the Gα subunit C terminus (Gα-CT) that are attached to chromatography beads (Gα-CT resin). Because the Gα-CT peptides bind active GPCRs with high affinity, the Gα-CT resin selectively purifies only active functional receptors. We use this method to purify both rhodopsin and the ß2-adrenergic receptor and show they can be purified in either active conformations or inactive conformations, simply by varying elution conditions. While simple in concept-leveraging the conserved GPCR-Gα-CT binding interaction for the purpose of GPCR purification-we think this approach holds excellent potential to isolate functional receptors for a myriad of uses, from structural biology to proteomics.

Styrene-maleic acid copolymer effects on the function of the GPCR rhodopsin in lipid nanoparticles.

Styrene-maleic acid (SMA) copolymers solubilize biological membranes to form lipid nanoparticles (SMALPs) that contain membrane proteins surrounded by native lipids, thus enabling the use of a variety of biophysical techniques for structural and functional studies. The question of whether SMALPs provide a truly natural environment or SMA solubilization affects the functional properties of membrane proteins, however, remains open. We address this question by comparing the photoactivation kinetics of rhodopsin, a G-protein-coupled receptor in the disk membranes of rod cells, in native membrane and SMALPs prepared at different molar ratios between SMA(3:1) and rhodopsin. Time-resolved absorption spectroscopy combined with complex kinetic analysis reveals kinetic and mechanistic differences between the native membrane and SMA-stabilized environment. The results suggest a range of molar ratios for nanoparticles suitable for kinetic studies.

Functional integrity of membrane protein rhodopsin solubilized by styrene-maleic acid copolymer.

Membrane proteins often require solubilization to study their structure or define the mechanisms underlying their function. In this study, the functional properties of the membrane protein rhodopsin in its native lipid environment were investigated after being solubilized with styrene-maleic acid (SMA) copolymer. The static absorption spectra of rhodopsin before and after the addition of SMA were recorded at room temperature to quantify the amount of membrane protein solubilized. The samples were then photobleached to analyze the functionality of rhodopsin upon solubilization. Samples with low or high SMA/rhodopsin ratios were compared to find a threshold in which the maximal amount of active rhodopsin was solubilized from membrane suspensions. Interestingly, whereas the highest SMA/rhodopsin ratios yielded the most solubilized rhodopsin, the rhodopsin produced under these conditions could not reach the active (Meta II) state upon photoactivation. The results confirm that SMA is a useful tool for membrane protein research, but SMA added in excess can interfere with the dynamics of protein activation.

Novel fluorescent GPCR biosensor detects retinal equilibrium binding to opsin and active G protein and arrestin signaling conformations.

Rhodopsin is a canonical class A photosensitive G protein-coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of ß-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.

Visualizing endogenous opioid receptors in living neurons using ligand-directed chemistry.

Identifying neurons that have functional opioid receptors is fundamental for the understanding of the cellular, synaptic and systems actions of opioids. Current techniques are limited to post hoc analyses of fixed tissues. Here we developed a fluorescent probe, naltrexamine-acylimidazole (NAI), to label opioid receptors based on a chemical approach termed 'traceless affinity labeling'. In this approach, a high affinity antagonist naltrexamine is used as the guide molecule for a transferring reaction of acylimidazole at the receptor. This reaction generates a fluorescent dye covalently linked to the receptor while naltrexamine is liberated and leaves the binding site. The labeling induced by this reagent allowed visualization of opioid-sensitive neurons in rat and mouse brains without loss of function of the fluorescently labeled receptors. The ability to locate endogenous receptors in living tissues will aid considerably in establishing the distribution and physiological role of opioid receptors in the CNS of wild type animals.

Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex.

Cannabis elicits its mood-enhancing and analgesic effects through the cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR) that signals primarily through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Activation of CB1-Gi signaling pathways holds potential for treating a number of neurological disorders and is thus crucial to understand the mechanism of Gi activation by CB1. Here, we present the structure of the CB1-Gi signaling complex bound to the highly potent agonist MDMB-Fubinaca (FUB), a recently emerged illicit synthetic cannabinoid infused in street drugs that have been associated with numerous overdoses and fatalities. The structure illustrates how FUB stabilizes the receptor in an active state to facilitate nucleotide exchange in Gi. The results compose the structural framework to explain CB1 activation by different classes of ligands and provide insights into the G protein coupling and selectivity mechanisms adopted by the receptor.

Single Proteoliposome High-Content Analysis Reveals Differences in the Homo-Oligomerization of GPCRs.

G-protein-coupled receptors (GPCRs) control vital cellular signaling pathways. GPCR oligomerization is proposed to increase signaling diversity. However, many reports have arrived at disparate conclusions regarding the existence, stability, and stoichiometry of GPCR oligomers, partly because of cellular complexity and ensemble averaging of intrareconstitution heterogeneities that complicate the interpretation of oligomerization data. To overcome these limitations, we exploited fluorescence-microscopy-based high-content analysis of single proteoliposomes. This allowed multidimensional quantification of intrinsic monomer-monomer interactions of three class A GPCRs (ß2-adrenergic receptor, cannabinoid receptor type 1, and opsin). Using a billion-fold less protein than conventional assays, we quantified oligomer stoichiometries, association constants, and the influence of two ligands and membrane curvature on oligomerization, revealing key similarities and differences for three GPCRs with decidedly different physiological functions. The assays introduced here will assist with the quantitative experimental observation of oligomerization for transmembrane proteins in general.

Purification of Functional CB1 and Analysis by Site-Directed Fluorescence Labeling Methods.

The human cannabinoid receptor, CB1, has been difficult to purify in a functional form, hampering structural and biophysical studies. Here, we present our approaches for obtaining pure, detergent solubilized, functional CB1. We also discuss our site-directed fluorescence labeling (SDFL) methods for identifying different structural changes that CB1 can undergo upon binding different cannabinoid ligands. To identify optimal CB1 constructs for these studies (those with the best expression levels, solubility in detergent and function), we first screened various CB1-green fluorescent protein chimeras in a mammalian expression system. Once identified, we then tagged the best candidates with the 1D4 epitope (the C-terminus of rhodopsin) and purified them using a single-step immunoaffinity process. The resulting, highly pure proteins retain their ability to activate G-protein, and are ~85% functional, as assessed by radioligand binding studies. The SDFL studies involve introducing single cysteine residues at key places in the receptor, then labeling them with a small fluorophore, bimane. The spectral properties of the bimane probe are then monitored before and after addition of cannabinoid ligands. Changes in fluorescence of the attached probe indicate regions of the receptor undergoing conformational changes upon ligand binding. Together, these approaches set the stage for a deeper understanding of the structure and function of CB1. Access to pure, functional CB1 makes subsequent structural studies possible (such as crystallography and single-particle EM analysis), and the SDFL studies enable a better structural and mechanistic understanding of this key receptor and the dynamic changes it undergoes during activation and attenuation.

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state.

Here, we describe two insights into the role of receptor conformational dynamics during agonist release (all-trans retinal, ATR) from the visual G protein-coupled receptor (GPCR) rhodopsin. First, we show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, we observe that this equilibrium can be shifted by either promoting the active-like population or increasing the agonist concentration. Second, we find that during decay of the signaling state an active-like, yet empty, receptor conformation can transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion is based on time-resolved, site-directed fluorescence labeling experiments that show a small, but reproducible, lag between the retinal leaving the protein and return of transmembrane helix 6 (TM6) to the inactive conformation, as determined from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissociation, either by addition of hydroxylamine or introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations show that rhodopsin can bind its agonist in equilibrium like a traditional GPCR, provide evidence that an active GPCR conformation can persist even after agonist release, and raise the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.

Biosensor reveals multiple sources for mitochondrial NAD⁺.

Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations.

Evidence that the Rhodopsin Kinase (GRK1) N-Terminus and the Transducin Gα C-Terminus Interact with the Same "Hydrophobic Patch" on Rhodopsin TM5.

Phosphorylation of G protein-coupled receptors (GPCRs) terminates their ability to couple with and activate G proteins by increasing their affinity for arrestins. Unfortunately, detailed information regarding how GPCRs interact with the kinases responsible for their phosphorylation is still limited. Here, we purified fully functional GPCR kinase 1 (GRK1) using a rapid method and used it to gain insights into how this important kinase interacts with the GPCR rhodopsin. Specifically, we find that GRK1 uses the same site on rhodopsin as the transducin (Gt) Gtα C-terminal tail and the arrestin "finger loop", a cleft formed in the cytoplasmic face of the receptor upon activation. Our studies also show GRK1 requires two conserved residues located in this cleft (L226 and V230) that have been shown to be required for Gt activation due to their direct interactions with hydrophobic residues on the Gα C-terminal tail. Our data and modeling studies are consistent with the idea that all three proteins (Gt, GRK1, and visual arrestin) bind, at least in part, in the same site on rhodopsin and interact with the receptor through a similar hydrophobic contact-driven mechanism.

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins.

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.

Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1.

G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1-it simultaneously increases agonist binding, decreases G--protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.

Conformational selection and equilibrium governs the ability of retinals to bind opsin.

Despite extensive study, how retinal enters and exits the visual G protein-coupled receptor rhodopsin remains unclear. One clue may lie in two openings between transmembrane helix 1 (TM1) and TM7 and between TM5 and TM6 in the active receptor structure. Recently, retinal has been proposed to enter the inactive apoprotein opsin (ops) through these holes when the receptor transiently adopts the active opsin conformation (ops*). Here, we directly test this "transient activation" hypothesis using a fluorescence-based approach to measure rates of retinal binding to samples containing differing relative fractions of ops and ops*. In contrast to what the transient activation hypothesis model would predict, we found that binding for the inverse agonist, 11-cis-retinal (11CR), slowed when the sample contained more ops* (produced using M257Y, a constitutively activating mutation). Interestingly, the increased presence of ops* allowed for binding of the agonist, all-trans-retinal (ATR), whereas WT opsin showed no binding. Shifting the conformational equilibrium toward even more ops* using a G protein peptide mimic (either free in solution or fused to the receptor) accelerated the rate of ATR binding and slowed 11CR binding. An arrestin peptide mimic showed little effect on 11CR binding; however, it stabilized opsin · ATR complexes. The TM5/TM6 hole is apparently not involved in this conformational selection. Increasing its size by mutagenesis did not enable ATR binding but instead slowed 11CR binding, suggesting that it may play a role in trapping 11CR. In summary, our results indicate that conformational selection dictates stable retinal binding, which we propose involves ATR and 11CR binding to different states, the latter a previously unidentified, open-but-inactive conformation.

Distance mapping in proteins using fluorescence spectroscopy: tyrosine, like tryptophan, quenches bimane fluorescence in a distance-dependent manner.

Tryptophan-induced quenching of fluorophores (TrIQ) uses intramolecular fluorescence quenching to assess distances in proteins too small (<15 Å) to be easily probed by traditional Forster resonance energy transfer methods. A powerful aspect of TrIQ is its ability to obtain an ultrafast snapshot of a protein conformation, by identifying "static quenching" (contact between the Trp and probe at the moment of light excitation). Here we report new advances in this site-directed fluorescence labeling (SDFL) approach, gleaned from recent studies of T4 lysozyme (T4L). First, we show that like TrIQ, tyrosine-induced quenching (TyrIQ) occurs for the fluorophore bimane in a distance-dependent fashion, although with some key differences. The Tyr "sphere of quenching" for bimane (≤10 Å) is smaller than for Trp (≤15 Å, Cα-Cα distance), and the size difference between the quenching residue (Tyr) and control (Phe) differs by only a hydroxyl group. Second, we show how TrIQ and TyrIQ can be used together to assess the magnitude and energetics of a protein movement. In these studies, we placed a bimane (probe) and Trp or Tyr (quencher) on opposite ends of a "hinge" in T4L and conducted TrIQ and TyrIQ measurements. Our results are consistent with an ∼5 Å change in Cα-Cα distances between these sites upon substrate binding, in agreement with the crystal structures. Subsequent Arrhenius analysis suggests the activation energy barrier (Ea) to this movement is relatively low (∼1.5-2.5 kcal/mol). Together, these results demonstrate that TyrIQ, used together with TrIQ, significantly expands the power of quenching-based distance mapping SDFL studies.

Nanoscale high-content analysis using compositional heterogeneities of single proteoliposomes.

Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the ß2-adrenergic receptor using ∼10(9)-fold less protein than conventional assays.

Rhodopsin TM6 can interact with two separate and distinct sites on arrestin: evidence for structural plasticity and multiple docking modes in arrestin-rhodopsin binding.

Various studies have implicated the concave surface of arrestin in the binding of the cytosolic surface of rhodopsin. However, specific sites of contact between the two proteins have not previously been defined in detail. Here, we report that arrestin shares part of the same binding site on rhodopsin as does the transducin Gα subunit C-terminal tail, suggesting binding of both proteins to rhodopsin may share some similar underlying mechanisms. We also identify two areas of contact between the proteins near this region. Both sites lie in the arrestin N-domain, one in the so-called "finger" loop (residues 67-79) and the other in the 160 loop (residues 155-165). We mapped these sites using a novel tryptophan-induced quenching method, in which we introduced Trp residues into arrestin and measured their ability to quench the fluorescence of bimane probes attached to cysteine residues on TM6 of rhodopsin (T242C and T243C). The involvement of finger loop binding to rhodopsin was expected, but the evidence of the arrestin 160 loop contacting rhodopsin was not. Remarkably, our data indicate one site on rhodopsin can interact with multiple structurally separate sites on arrestin that are almost 30 Å apart. Although this observation at first seems paradoxical, in fact, it provides strong support for recent hypotheses that structural plasticity and conformational changes are involved in the arrestin-rhodopsin binding interface and that the two proteins may be able to interact through multiple docking modes, with arrestin binding to both monomeric and dimeric rhodopsin.

Fluorescence spectroscopy of rhodopsins: insights and approaches.

Fluorescence spectroscopy has become an established tool at the interface of biology, chemistry and physics because of its exquisite sensitivity and recent technical advancements. However, rhodopsin proteins present the fluorescence spectroscopist with a unique set of challenges and opportunities due to the presence of the light-sensitive retinal chromophore. This review briefly summarizes some approaches that have successfully met these challenges and the novel insights they have yielded about rhodopsin structure and function. We start with a brief overview of fluorescence fundamentals and experimental methodologies, followed by more specific discussions of technical challenges rhodopsin proteins present to fluorescence studies. Finally, we end by discussing some of the unique insights that have been gained specifically about visual rhodopsin and its interactions with affiliate proteins through the use of fluorescence spectroscopy. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

The membrane proximal region of the cannabinoid receptor CB1 N-terminus can allosterically modulate ligand affinity.

The human cannabinoid receptor, CB1, a G protein-coupled receptor (GPCR), contains a relatively long (∼110 a.a.) amino terminus, whose function is still not defined. Here we explore a potential role for the CB1 N-terminus in modulating ligand binding to the receptor. Although most of the CB1 N-terminus is not necessary for ligand binding, previous studies have found that mutations introduced into its conserved membrane proximal region (MPR) do impair the receptors ability to bind ligand. Moreover, within the highly conserved MPR (∼ residues 90-110) lie two cysteine residues that are invariant in all CB1 receptors. We find these two cysteines (C98 and C107) form a disulfide in heterologously expressed human CB1, and this C98-C107 disulfide is much more accessible to reducing agents than the previously known disulfide in extracellular loop 2 (EL2). Interestingly, the presence of the C98-C107 disulfide modulates ligand binding to the receptor in a way that can be quantitatively analyzed by an allosteric model. The C98-C107 disulfide also alters the effects of allosteric ligands for CB1, Org 27569 and PSNCBAM-1. Together, these results provide new insights into how the N-terminal MPR and EL2 act together to influence the high-affinity orthosteric ligand binding site in CB1 and suggest that the CB1 N-terminal MPR may be an area through which allosteric modulators can act.

A constitutively activating mutation alters the dynamics and energetics of a key conformational change in a ligand-free G protein-coupled receptor.

G protein-coupled receptors (GPCRs) undergo dynamic transitions between active and inactive conformations. Usually, these conversions are triggered when the receptor detects an external signal, but some so-called constitutively activating mutations, or CAMs, induce a GPCR to bind and activate G proteins in the absence of external stimulation, in ways still not fully understood. Here, we investigated how a CAM alters the structure of a GPCR and the dynamics involved as the receptor transitions between different conformations. Our approach used site-directed fluorescence labeling (SDFL) spectroscopy to compare opsin, the ligand-free form of the GPCR rhodopsin, with opsin containing the CAM M257Y, focusing specifically on key movements that occur in the sixth transmembrane helix (TM6) during GPCR activation. The site-directed fluorescence labeling data indicate opsin is constrained to an inactive conformation both in detergent micelles and lipid membranes, but when it contains the M257Y CAM, opsin is more dynamic and can interact with a G protein mimetic. Further study of these receptors using tryptophan-induced quenching (TrIQ) methods indicates that in detergent, the CAM significantly increases the population of receptors in the active state, but not in lipids. Subsequent Arrhenius analysis of the TrIQ data suggests that, both in detergent and lipids, the CAM lowers the energy barrier for TM6 movement, a key transition required for conversion between the inactive and active conformations. Together, these data suggest that the lowered energy barrier is a primary effect of the CAM on the receptor dynamics and energetics.