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Background: The growing evidence that G protein-coupled receptors (GPCRs) not only form oligomers but that the oligomers also may modulate the receptor function provides a promising avenue in the area of drug design. Highly selective drugs targeting distinct oligomeric sub-states offer the potential to increase efficacy while reducing side effects. In this regard, determining the various oligomeric configurations and geometric sub-states of a membrane receptor is of utmost importance. Methods: In this report, we have reviewed two techniques that have proven to be valuable in monitoring the quaternary structure of proteins in vivo: Fӧrster resonance energy transfer (FRET) spectrometry and fluorescence intensity fluctuation (FIF) spectrometry. In FRET spectrometry, distributions of pixel-level FRET efficiency are analyzed using theoretical models of various quaternary structures to determine the geometry and stoichiometry of protein oligomers. In FIF spectrometry, spatial fluctuations of fluorescent molecule intensities are analyzed to reveal quantitative information on the size and stability of protein oligomers. Results: We demonstrate the application of these techniques to a number of different fluorescence-based studies of cells expressing fluorescently labeled membrane receptors, both in the presence and absence of various ligands. The results show the effectiveness of using FRET spectrometry to determine detailed information regarding the quaternary structure receptors form, as well as FIF and FRET for determining the relative abundance of different-sized oligomers when an equilibrium forms between such structures. Conclusion: FRET and FIF spectrometry are valuable techniques for characterizing membrane receptor oligomers, which are of great benefit to structure‐based drug design.

Here, we introduce fluorescence intensity fluctuation spectrometry for determining the identity, abundance and stability of protein oligomers. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric states of the epidermal growth factor receptor and the secretin receptor in the presence and absence of their agonist ligands. This method is fast and is scalable for high-throughput screening of drugs targeting protein–protein interactions.

CXCR4, a member of the family of chemokine-activated G protein-coupled receptors, is widely expressed in immune response cells. It is involved in both cancer development and progression as well as viral infection, notably by HIV-1. A variety of methods, including structural information, have suggested that the receptor may exist as a dimer or an oligomer. However, the mechanistic details surrounding receptor oligomerization and its potential dynamic regulation remain unclear. Using both biochemical and biophysical means, we confirm that CXCR4 can exist as a mixture of monomers, dimers, and higher-order oligomers in cell membranes and show that oligomeric structure becomes more complex as receptor expression levels increase. Mutations of CXCR4 residues located at a putative dimerization interface result in monomerization of the receptor. Additionally, binding of the CXCR4 antagonist IT1t-a small drug-like isothiourea derivative-rapidly destabilizes the oligomeric structure, whereas AMD3100, another well-characterized CXCR4 antagonist, does not. Although a mutation that regulates constitutive activity of CXCR4 also results in monomerization of the receptor, binding of IT1t to this variant promotes receptor dimerization. These results provide novel insights into the basal organization of CXCR4 and how antagonist ligands of different chemotypes differentially regulate its oligomerization state.

The resistance of methicillin-resistant Staphylococcus aureus to antibiotics presents serious clinical problems that prompted the need for finding alternative or combination therapies. One such therapy is irradiation with blue light. To determine the alterations in metabolic processes implicated in the observed antimicrobial effects of blue light, we investigated the changes in membrane potential and the presence of free-radical-producing photo-acceptor molecules. Bacterial cultures irradiated with one or two doses of 405nm laser light (each consisting of 121J/cm2) were imaged with spectrally resolved laser-scanning microscopes to detect endogenous fluorescent species as well as the voltage sensitive dye 3,3'-Diethyloxacarbocyanine iodide. The endogenous fluorescence indicated the presence of photosensitizers (i.e., porphyrins, NADH, FAD) in the cells, while the exogenous signal allowed us to monitor rapid changes in transmembrane potential following treatment with light. The changes were drastic within the first 5min after irradiation with the first dose and continued slowly after the second irradiation. These results suggest that the early antimicrobial activity of blue light results from alteration of membrane integrity with a consequent decrease in membrane polarization and rapid alteration of vital cellular functions. The observation of an early antimicrobial activity of light is very encouraging, as it suggests that treatment does not necessarily have to be administered over a long period of time.

Using radiofrequency dielectric spectroscopy, we have investigated the impact of the interaction between a G protein-coupled receptor (GPCR), the sterile2 α-factor receptor protein (Ste2), and its cognate agonist ligand, the α-factor pheromone, on the dielectric properties of the plasma membrane in living yeast cells (Saccharomyces cerevisiae). The dielectric properties of a cell suspension containing a saturating concentration of α-factor were measured over the frequency range 40Hz–110 MHz and compared to the behavior of a similarly prepared suspension of cells in the absence of α-factor. A spherical three-shell model was used to determine the electrical phase parameters for the yeast cells in both types of suspensions. The relative permittivity of the plasma membrane showed a significant increase after exposure to α-factor (by 0.06 ± 0.05). The equivalent experiment performed on yeast cells lacking the ability to express Ste2 showed no change in plasma membrane permittivity. Interestingly, a large change also occurred to the electrical properties of the cellular interior after the addition of α-factor to the cell suspending medium, whether or not the cells were expressing Ste2. We present a number of different complementary experiments performed on the yeast to support these dielectric data and interpret the results in terms of specific cellular reactions to the presence of α-factor.

Current technologies for probing membrane protein association and stability in cells are either very laborious or lack the bandwidth needed for fully quantitative analysis. Here we introduce a platform, termedone-ortwo-dimensional fluorescence intensity fluctuation spectrometry, for determining the identity, abundance, and stability of oligomers of differing sizes. The sensitivity of this approach is demonstrated by using monomers and oligomers of known sizes in both solutions and cell membranes. The analysis was extended to uncover the oligomeric states and their stability for both the epidermal growth factor receptor, a receptor tyrosine kinase, and the G protein-coupled secretin receptor. In both cases, agonist ligand binding shifted the equilibrium from monomers or dimers to rather large oligomers. Our method can be used in conjunction with a variety of light-based microscopy techniques, is several orders of magnitude faster than current approaches, and is scalable for high-throughput analyses.

Here we introduce the fully quantified spectral imaging (FSI) method as a new tool to probe the stoichiometry and stability of protein complexes in biological membranes. The FSI method yields two dimensional membrane concentrations and FRET efficiencies in native plasma membranes. It can be used to characterize the association of membrane proteins: to differentiate between monomers, dimers, or oligomers, to produce binding (association) curves, and to measure the free energies of association in the membrane. We use the FSI method to study the lateral interactions of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a member of the receptor tyrosine kinase (RTK) superfamily, in plasma membranes, in vivo. The knowledge gained through the use of the new method challenges the current understanding of VEGFR2 signaling.

Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The method provides highly resolved spectra of molecules or molecular complexes at each image pixel, and it does so on a timescale shorter than that of molecular diffusion, which scrambles the spectral information. Acquisition of an entire spectrally resolved image, however, is slower than that of broad-bandwidth microscopes because it takes longer times to collect the same number of photons at each emission wavelength as in a broad bandwidth. Here, we demonstrate an optical micro-spectroscopic scheme that employs a laser beam shaped into a line to excite in parallel multiple sample voxels. The method presents dramatically increased sensitivity and/or acquisition speed and, at the same time, has excellent spatial and spectral resolution, similar to point-scan configurations. When applied to FRET imaging using an oligomeric FRET construct expressed in living cells and consisting of a FRET acceptor linked to three donors, the technique based on line-shaped excitation provides higher accuracy compared to the point-scan approach, and it reduces artifacts caused by photobleaching and other undesired photophysical effects.

Transmembrane proteins known as G protein-coupled receptors (GPCRs) have been shown to form functional homo- or hetero-oligomeric complexes, although agreement has been slow to emerge on whether homo-oligomerization plays functional roles. Here we introduce a platform to determine the identity and abundance of differing quaternary structures formed by GPCRs in living cells following changes in environmental conditions, such as changes in concentrations. The method capitalizes on the intrinsic capability of FRET spectrometry to extract oligomer geometrical information from distributions of FRET efficiencies (or FRET spectrograms) determined from pixel-level imaging of cells, combined with the ability of the statistical ensemble approaches to FRET to probe the proportion of different quaternary structures (such as dimers, rhombus or parallelogram shaped tetramers, etc.) from averages over entire cells. Our approach revealed that the yeast pheromone receptor Ste2 forms predominantly tetramers at average expression levels of 2 to 25 molecules per pixel (2.8 · 10 − 6 to 3.5 · 10 − 5 molecules/nm 2 ), and a mixture of tetramers and octamers at expression levels of 25–100 molecules per pixel (3.5 · 10 − 5 to 1.4 · 10 − 4 molecules/nm 2 ). Ste2 is a class D GPCR found in the yeast Saccharomyces cerevisiae of the mating type a , and binds the pheromone α-factor secreted by cells of the mating type α . Such investigations may inform development of antifungal therapies targeting oligomers of pheromone receptors. The proposed FRET imaging platform may be used to determine the quaternary structure sub-states and stoichiometry of any GPCR and, indeed, any membrane protein in living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that initiates phototransduction in the retina. The receptor consists of the apoprotein opsin covalently linked to the inverse agonist 11-cis retinal. Rhodopsin and opsin have been shown to form oligomers within the outer segment disc membranes of rod photoreceptor cells. However, the physiological relevance of the observed oligomers has been questioned since observations were made on samples prepared from the retina at low temperatures. To investigate the oligomeric status of opsin in live cells at body temperatures, we utilized a novel approach called Förster resonance energy transfer spectrometry, which previously has allowed the determination of the stoichiometry and geometry (i.e. quaternary structure) of various GPCRs. In the current study, we have extended the method to additionally determine whether or not a mixture of oligomeric forms of opsin exists and in what proportion. The application of this improved method revealed that opsin expressed in live Chinese hamster ovary (CHO) cells at 37°C exists as oligomers of various sizes. At lower concentrations, opsin existed in an equilibrium of dimers and tetramers. The tetramers were in the shape of a near-rhombus. At higher concentrations of the receptor, higher-order oligomers began to form. Thus, a mixture of different oligomeric forms of opsin is present in the membrane of live CHO cells and oligomerization occurs in a concentration-dependent manner. The general principles underlying the concentration-dependent oligomerization of opsin may be universal and apply to other GPCRs as well.

Forster Resonance Energy Transfer (FRET) from an electronically excited donor to an acceptor molecule is used to quantify the extent of interactions between molecules. For example, FRET can be utilized to determine how proteins form complexes by tagging them differentially with donor and acceptor fluorophores. Recent advances in the FRET theory combined with a novel spectrally resolved two-photon microscope have strengthened the effectiveness of the FRET technique and have enabled us to determine the size and structure of protein oligomers in living cells (Raicu et al., Nature Photonics, 3, 2009). In an ongoing research project in our lab, we are are probing the movement of protomers in an oligomer by appropriately tagging and systematically altering a fluorophore position within each protomer. In the work described here, we show the application of this method in yeast cells (S. cerevisiae) that express the sterile 2 alpha factor protein (Ste2p, a G protein-coupled receptor) tagged with two different variants of the green fluorescent protein (GFP). Previously, we showed by tagging GFP2/YPF (donor/acceptor fluorophores) variants at the C-terminus of Ste2p that Ste2p is self-assembled into a rhombus shaped tetramer. The measured FRET efficiencies of the Ste2p oligomers, which were tagged with fluorophores at various locations in the Ste2p amino acid sequence, were calibrated against FRET reference standards in order to extract information regarding the relative orientations of the fluorescent tags. The effect of an agonist, the yeast mating pheromone alpha factor, on the measured FRET efficiencies was quantified and compared for the various fluorescent tag locations to reveal information regarding the relative movement of the Ste2p protomer segments upon binding of the agonist.