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.

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.

Functional independence of endogenous μ- and δ-opioid receptors co-expressed in cholinergic interneurons.

Class A G-protein-coupled receptors (GPCRs) normally function as monomers, although evidence from heterologous expression systems suggests that they may sometimes form homodimers and/or heterodimers. This study aims to evaluate possible functional interplay of endogenous µ- and δ-opioid receptors (MORs and DORs) in mouse neurons. Detecting GPCR dimers in native tissues, however, has been challenging.

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.

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.

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'.

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 G. Activation of CB1-G signaling pathways holds potential for treating a number of neurological disorders and is thus crucial to understand the mechanism of G activation by CB1. Here, we present the structure of the CB1-G 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.

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.

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

The human cannabinoid receptor, CB, 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 CB. We also discuss our site-directed fluorescence labeling (SDFL) methods for identifying different structural changes that CB can undergo upon binding different cannabinoid ligands.

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.

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.

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.

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.

The Enzymatic Activity of Lipases Correlates with Polarity-Induced Conformational Changes: A Trp-Induced Quenching Fluorescence Study.

Triacylglycerol hydrolases (EC 3.1.1.

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*).

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).

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.

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.

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.

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.

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.

A key agonist-induced conformational change in the cannabinoid receptor CB1 is blocked by the allosteric ligand Org 27569.

Allosteric ligands that modulate how G protein-coupled receptors respond to traditional orthosteric drugs are an exciting and rapidly expanding field of pharmacology. An allosteric ligand for the cannabinoid receptor CB1, Org 27569, exhibits an intriguing effect; it increases agonist binding, yet blocks agonist-induced CB1 signaling. Here we explored the mechanism behind this behavior, using a site-directed fluorescence labeling approach.

Rhodopsin in nanodiscs has native membrane-like photointermediates.

Time-dependent studies of membrane protein function are hindered by extensive light scattering that impedes application of fast optical absorbance methods. Detergent solubilization reduces light scattering but strongly perturbs rhodopsin activation kinetics. Nanodiscs may be a better alternative if they can be shown to be free from the serious kinetic perturbations associated with detergent solubilization.

What site-directed labeling studies tell us about the mechanism of rhodopsin activation and G-protein binding.

Rhodopsin is the photoreceptor protein responsible for dim-light vision in mammals. Due to extensive biophysical, structural and computational analysis of this membrane protein, it is presently the best understood G-protein coupled receptor (GPCR). Here I briefly review one approach that has been extensively used to identify dynamic and structural changes in rhodopsin--the use of site-directed labeling methods (SDL) coupled with electron paramagnetic resonance (EPR) and fluorescence spectroscopy.