Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor1

Background information. The idea that GPCRs (G‐protein‐coupled receptors) may exist as homo‐ or hetero‐oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM₁ (human M₁ muscarinic acetylcholine receptor subtype). Results. We analysed the hM₁ oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native‐PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO‐M1 cells (Chinese‐hamster ovary cells expressing muscarinic M₁ receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M₁ receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M₁ receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor. Conclusions. Our results suggest that MT7 binds to a dimeric form of hM₁ receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre‐existing muscarinic receptor homodimers.     

A vinblastine fluorescent probe for pregnane X receptor in a time-resolved fluorescence resonance energy transfer assay

The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows the binding of many drugs and drug leads to it, possibly causing undesired drug–drug interactions. Therefore, it is crucial to evaluate whether lead compounds bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. One fluorescent PXR probe is currently commercially available; however, because its chemical structure is not publicly disclosed, it is not optimal for studying ligand–PXR interactions. Here we report the characterization of BODIPY FL–vinblastine, generated by labeling vinblastine with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), as a high-affinity ligand for human PXR with a K_d value of 673 nM. We provide evidence that BODIPY FL–vinblastine is a unique chemical entity different from either vinblastine or the fluorophore BODIPY FL in its function as a high-affinity human PXR ligand. We describe a BODIPY FL–vinblastine-based human PXR time-resolved fluorescence resonance energy transfer assay, which was used to successfully test a panel of human PXR ligands. The BODIPY FL–vinblastine-based biochemical assay is suitable for high-throughput screening to evaluate whether lead compounds bind to PXR.     

Fluorescence resonance energy transfer between bovine serum albumin and fluoresceinamine

Physical binding‐mediated organic dye direct‐labelling of proteins could be a promising technology for bio‐nanomedical applications. Upon binding, it was found that fluorescence resonance energy transfer (FRET) occurred between donor bovine serum albumin (BSA; an amphiphilic protein) and acceptor fluoresceinamine (FA; a hydrophobic fluorophore), which could explain fluorescence quenching found for BSA. FRET efficiency and the distance between FA and BSA tryptophan residues were determined to 17% and 2.29 nm, respectively. Using a spectroscopic superimposition method, the saturated number of FAs that bound to BSA was determined as eight to give a complex formula of FA₈–BSA. Finally, molecular docking between BSA and FA was conducted, and conformational change that occurred in BSA upon binding to FA molecules was also studied by three‐dimensional fluorescence microscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

Alternative splicing of RGS8 gene changes the binding property to the M1 muscarinic receptor to confer receptor type-specific Gq regulation

RGS proteins constitute a large family that modulates heterotrimeric G-protein signaling. We previously showed that RGS8 suppressed Gq signaling in a receptor type-specific manner. To elucidate molecular mechanisms underlying receptor-specific attenuation by RGS8, we examined whether RGS8 can interact with certain G-protein-coupled receptors. By pull-down assay, we showed that RGS8 directly binds to the third intracellular (i3) loop of M1 and M3 muscarinic acetylcholine receptors (mAChRs). The binding of RGS8S, a splice variant with a different N-terminus, was weaker. RGS8 could bind specifically to the C-terminal part of M1i3 (containing amino acids of 304-353 of i3 of human M1-mAChR), but RGS8S could not. Moreover, deletion of the N-terminal 9 amino acids and substitution of both Arg-8 and Arg-9 of RGS8 with Ala resulted in reduced binding to M1i3. BRET experiments revealed that RGS8 actually interacts with M1-mAChR, but RGS8S does not interact in the living cells. The RGS8 mutant, which had less binding ability to M1i3, showed a reduced inhibitory function of Gq signaling through M1-mAChR. These results demonstrated that RGS8 can directly interact with M1-mAChR via its N-terminus and the i3 loop of the receptor, and this binding must play an essential role in receptor-specific suppression by RGS8.     

Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells

A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100 Å of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.     

Fluorescence resonance energy transfer between two quantum dots with immunocomplexes of antigen and antibody as a bridge.

In this study, 573 nm quantum dots (QDs)-rabbit IgG-goat anti-rabbit IgG-638 nm QDs immunocomplexes were prepared, utilizing antigen-antibody interaction. 573 nm-emitting QDs were conjugated to antigen (rabbit IgG) and 638 nm-emitting QDs were conjugated to antibody (goat anti-rabbit IgG) via electrostatic/hydrophilic self-assembly, respectively. The mutual affinity of the antigen and antibody brought two kinds of QDs close enough to result in fluorescence resonance energy transfer (FRET) between them; the luminescence emission of 573 nm QDs was quenched, while that of 638 nm QDs was enhanced. The luminescence emission of 573 nm QDs could be recovered when the immunocomplexes were exposed to the unlabelled rabbit IgG antigen. The FRET efficiency (E) and the distance between the donor and the acceptor were calculated.     

Fluorescence resonance energy transfer between donor-acceptor pair on two oligonucleotides hybridized adjacently to DNA template

We use fluorescein as the energy donor and rhodamine as the acceptor to measure the efficiency of fluorescence resonance energy transfer (FRET) in a set of hybridized DNA constructs. The two fluorophores are covalently attached via linkers to two separate oligonucleotides with fluorescein at the 3' end of one oligonucleotide and rhodamine at the 5' end or in the middle of another nucleotide. For the FRET analysis both fluorophore-labeled oligonucleotides are hybridized to adjacent sections of the same DNA template to form a three-component duplex with a one base gap between the two labeled oligonucleotides. A similar configuration is implemented for a quantitative real-time polymerase chain reaction (PCR) with LightCycler technology, where a 1-5 base separation between donor and acceptor is recommended to optimize energy transfer efficiencies. Our constructs cover donor-acceptor separations from 2 to 17 base pairs (approximately 10-70 A). The results show that, when the two fluorophores are located at close distances (less than 8 base separation), FRET efficiencies are above 80%, although there may be ground-state interactions between fluorophores when the separation is under about 6 bases. Modeling calculations are used to predict the structure of these three-component constructs. The duplex mostly retains a normal double helical structure, although slight bending may occur near the unpaired base in the DNA template. Stable and reproducible energy transfer is also observed over the distance range investigated here in real-time thermal cycling. The study identifies important parameters that determine FRET response in applications such as real-time PCR.     

RecQ helicase-catalyzed DNA unwinding detected by fluorescence resonance energy transfer

A fluorometric assay was used to study the DNA unwinding kinetics induced by Escherichiacoli RecQ helicase.This assay was based on fluorescence resonance energy transfer and carried out onstopped-flow,in which DNA unwinding was monitored by fluorescence emission enhancement of fluoresceinresulting from helicase-catalyzed DNA unwinding.By this method,we determined the DNA unwinding rateof RecQ at different enzyme concentrations.We also studied the dependences of DNA unwinding magnitudeand rate on magnesium ion concentration.We showed that this method could be used to determine thepolarity of DNA unwinding.This assay should greatly facilitate further study of the mechanism for RecQ-catalyzed DNA unwinding.     

生物学中荧光共振能量转移的研究应用进展

荧光共振能量转移（FRET）可用于对生物大分子之间的距离进行定性、定量检测,所采用的材料、方法在近年都有了很大的发展,在核酸、蛋白质、细胞器结构功能检测、免疫测定、配体－受体相互作用测定等方面都有巧妙而有效的应用,应用前景十分广阔。     

From structure to clinic: Design of a muscarinic M1 receptor agonist with the potential to treat Alzheimer’s disease

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Intracellular interaction between syntaxin and Munc 18-1 revealed by fluorescence resonance energy transfer

Neurosecretion is catalyzed by assembly of a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-complex composed of SNAP-25, synaptobrevin and syntaxin. Munc 18-1 is known to bind to syntaxin in vitro. This interaction prevents assembly of the SNARE-complex, but might also affect intracellular targeting of the proteins. We have fused syntaxin and Munc 18 to the yellow- (YFP) or cyan-fluorescence-protein (CFP) and expressed the constructs in CHO- and MDCK-cells. We have studied their localization with confocal microscopy and a possible protein-protein interaction with fluorescence-resonance energy transfer (FRET). YFP-syntaxin localizes to intracellular membranes. CFP-Munc 18 is present in the cytoplasm as expected for a protein lacking membrane targeting domains. However, Munc 18 is redirected to internal membranes when syntaxin is coexpressed, but only limited transport of the proteins to the plasma membrane was observed. An interaction between Munc 18 and syntaxin could be demonstrated by FRET using two methods, sensitized acceptor fluorescence and acceptor photobleaching. A mutation in syntaxin (L165A, E166A), which is known to inhibit binding to Munc 18 in vitro, prevents colocalization of the proteins and also the FRET signal. Thus, a protein-protein interaction between Munc 18 and syntaxin occurs on intracellular membranes, which is required but not sufficient for quantitative transport of both proteins to the plasma membrane.     

Multiplexed DNA detection using a gold nanorod‐based fluorescence resonance energy transfer technique

A fluorescence resonance energy transfer method for multiplex detection DNA based on gold nanorods had been successfully constructed. This method is simple, easy to operate, good selectivity, no requirement to label the probe molecule and can analyze simultaneously multiple targets of DNA in one sample. The limit of detection for the 18‐mer, 27‐mer and 30‐mer targets is 0.72, 1.0 and 0.43 nM at a signal‐to‐noise ratio of 3. The recoveries of three targets were 96.57–98.07%, 99.12–100.04% and 97.29–99.93%, respectively. The results show that the method can be used to analyze a clinical sample or a biological sample; it also can be used to develop new probes for rapid, sensitive and highly selective multiplex detection of analytes in real samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Conformations within soluble oligomers and insoluble aggregates revealed by resonance energy transfer

A fluorescently labeled 20‐residue polyglutamic acid (polyE) peptide 20 amino acid length polyglutamic acid (E₂₀) was used to study structural changes which occur in E₂₀ as it co‐aggregates with other unlabeled polyE peptides. Resonance energy transfer (RET) was performed using an o‐aminobenzamide donor at the N‐terminus and 3‐nitrotyrosine acceptor at the C‐terminus of E₂₀. PolyE aggregates were not defined as amyloid, as they were nonfibrillar and did not bind congo red. Circular dichroism measurements indicate that polyE aggregation involves a transition from α‐helical monomers to aggregated β‐sheets. Soluble oligomers are also produced along with aggregates in the reaction, as determined through size exclusion chromatography. Time‐resolved and steady‐state RET measurements reveal four dominant E₂₀ conformations: (1) a partially collapsed conformation (24 Å donor–acceptor distance) in monomers, (2) an extended conformation in soluble oligomers (>29 Å donor–acceptor distance), (3) a minor partially collapsed conformation (22 Å donor‐acceptor distance) in aggregates, and (4) a major highly collapsed conformation (13 Å donor–acceptor distance) in aggregates. These findings demonstrate the use of RET as a means of determining angstrom‐level structural details of soluble oligomer and aggregated states of proteins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 299–317, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Photobleaching fluorescence resonance energy transfer reveals ligand-induced oligomer formation of human somatostatin receptor subtypes

The existence of dimers and higher oligomers of G-protein-coupled receptors (GPCRs) has been frequently reported using strategies based on coimmunoprecipitation or Western blot assays. These methods rely on highly artificial systems with overexpressed receptors, resulting in conflicting observations on the question of whether GPCR dimers are preformed or are formed in response to agonist treatment. Fluorescence resonance energy transfer (FRET) microscopy is a superior and less perturbing technique which can be performed on selected cell regions, e.g., plasma membrane of intact cells with a sensitivity high enough to allow study under physiological levels of receptor expression. Here we describe the application of photobleaching (pb) FRET microscopy for investigating ligand-dependent oligomerization of somatostatin receptors. Procedures for the introduction of suitable donor-acceptor fluorophores in a given GPCR are described. The competitive nature of FRET and photobleaching is exploited to enable the indirect measurement of FRET via its effect on donor photobleaching lifetimes on a pixel-by-pixel basis. The method allows enhanced resolution between 10 and 100A and represents a sensitive and specific biophysical tool for characterizing the assembly and regulation of GPCR oligomers on the cell surface.     

Highly selective detection of deoxyribonucleic acid in living cells using RecA-green fluorescent protein-single-stranded deoxyribonucleic acid filament fluorescence resonance energy transfer probe

A fluorescence resonance energy transfer (FRET) method was developed for double-stranded deoxyribonucleic acid (dsDNA) detection in living cells using the RecA-GFP (green fluorescent protein) fusion protein filament. In brief, the thiol-modified single-stranded DNA (ssDNA) was attached to gold nanoparticles (AuNPs); on the contrary, the prepared RecA-GFP fusion protein interacted with ssDNA. Due to the FRET between AuNPs and RecA-GFP, fluorescence of RecA-GFP fusion protein was quenched. In the presence of homologous dsDNA, homologous recombination occurred to release RecA-GFP fusion protein. Thus, the fluorescence of RecA-GFP was recovered. The dsDNA concentration was detected using fluorescence intensity of RecA-GFP. Under optimal conditions, this method could detect dsDNA activity as low as 0.015 optical density (OD) Escherichia coli cells, with a wide linear range from 0.05 to 0.9 OD cells, and the regression equation was ΔF = 342.7c + 78.9, with a linear relationship coefficient of 0.9920. Therefore, it provided a promising approach for the selective detection of dsDNA in living cells for early clinical diagnosis of genetic diseases.     

Preparation and characterization of Alexa Fluor 594-labeled epidermal growth factor for fluorescence resonance energy transfer studies: application to the epidermal growth factor receptor

We have prepared and characterized a new fluorescent derivative of murine epidermal growth factor (EGF), Alexa Fluor 594-labeled EGF (A-EGF), for fluorescence studies of EGF-EGF receptor interactions. We describe the synthesis of this derivative and its physical and biological characterization. The significant overlap between the excitation and the emission spectra of A-EGF makes this probe well suited to fluorescence resonance energy homo-transfer. Using time-resolved fluorescence to examine the oligomeric state of the EGF receptor, we have observed resonance energy homo-transfer of A-EGF bound to EGF receptors in cells, but not of A-EGF bound to EGF receptors in membrane vesicles. Our results, interpreted in the context of recent crystallographic studies of the ligand-binding domains of EGF receptors, suggest that observed fluorescence resonance energy transfer does not result from transfer within receptor dimers, but rather results from transfer within higher-order oligomers. Furthermore, our results support a structural model for oligomerization of EGF receptors in which dimers are positioned head-to-head with respect to the ligand-binding site, consistent with the head-to-head interactions observed between adjacent receptor dimers by X-ray crystallography.     

Use of fluorescence resonance energy transfer to analyze oligomerization of G-protein-coupled receptors expressed in yeast

Oligomerization or dimerization of G-protein-coupled receptors (GPCRs) has emerged as an important theme in signal transduction. This concept has recently gained widespread interest due to the application of direct and noninvasive biophysical techniques such as fluorescence resonance energy transfer (FRET), which have shown unequivocally that several types of GPCR can form dimers or oligomers in living cells. Current challenges are to determine which GPCRs can self-associate and/or interact with other GPCRs, to define the molecular principles that govern these specific interactions, and to establish which aspects of GPCR function require oligomerization. Although these questions ultimately must be addressed by using GPCRs expressed endogenously in their native cell types, analysis of GPCR oligomerization in heterologous expression systems will be useful to survey which GPCRs can interact, to conduct structure-function studies, and to identify peptides or small molecules that disrupt GPCR oligomerization and function. Here, we describe methods employing scanning fluorometry to detect FRET between GPCRs tagged with enhanced cyan and yellow fluorescent proteins (CFP and YFP) in living yeast cells. This approach provides a powerful means to analyze oligomerization of a variety of GPCRs that can be expressed in yeast, such as adrenergic, adenosine, C5a, muscarinic acetylcholine, vasopressin, opioid, and somatostatin receptors.     

Fluorescence and energy transfer in phycobiliprotein-containing algae at low temperature

Fluorescence emission spectra of Anacystis nidulans, Porphyridium cruentum and Cyanidium caldarium, three phycobiliprotein-containing algae, were measured at temperatures between 4 and 120 K in the absence and in the presence of quinones as quenchers of chlorophyll fluorescence. In all species three major emission bands were observed in the chlorophyll a region, near 685 nm (F-685), 695 nm (F-695) and between 710 and 730 nm. Additional bands were observed at shorter wavelengths; these were preferentially excited by light absorbed by the phycobiliproteins and are presumably due to phycocyanins and allophycocyanins.

The amplitudes of F-685, F-695 and the long-wave emission showed a distinct increase upon cooling. For F-685 and F-695 the temperature dependence was similar to that earlier observed with spinach chloroplasts, for the long-wave emission it appeared to depend on the location of the emission bands, which was different for different species. All three bands were strongly quenched by quinones. These and other data suggest that the origin of these bands is the same as in higher plants, and that the fluorescence increase upon cooling can be explained by a lowering of the efficiency of energy transfer between chlorophyll molecules. It is concluded that at most a small percentage of the emission at 685 nm can be ascribed to allophycocyanin B, and that the efficiency of energy transfer between allophycocyanin B and chlorophyll a probably exceeds 99% both at 77 and 4 K. Experiments with isolated phycobilisomes suggest that energy transfer from allophycocyanin to allophycocyanin B occurs with an efficiency of about 90% at low temperature.

The effect of quenchers can be understood by the assumption that the quenching is caused by the formation of non-fluorescent traps in the bulk chlorophyll. Of three quinones tested, the strongest quenching was observed with dibromothymoquinone, which quenched F-685, F-695 and the long-wave emission approximately equally. Menadione and 1,4-naphthoquinone, however, preferentially quenched the long-wave bands, indicating a stronger interaction with Photosystem I than with Photosystem II chlorophylls.