Homogeneous TR-FRET high-throughput screening assay for calcium-dependent multimerization of sorcin

A homogeneous high-throughput screening method based on time-resolved fluorescence resonance energy transfer (TR-FRET) for the measurement of calcium-dependent multimerization of an EF-hand protein, sorcin, is described. The assay is based on a specific sorcin binding peptide conjugated either with an intrinsically highly fluorescent europium chelate (donor) or an Alexa Fluor 700 fluorophore (acceptor). Addition of calcium results in multimerization of sorcin, allowing several peptides to bind simultaneously to the epitopes of the multimeric protein complex, and the proximity of peptides labeled either with donor or acceptor label results in fluorescence resonance energy transfer between the 2 labels. When no calcium is present, the protein remains in a monomer form, and thus no FRET can take place. In the optimized assay construct, the assay was performed in 45 min, and a more than 20-fold signal-to-background ratio was achieved. The reversibility of sorcin multimerization was shown by chelating free calcium with ethylenediamine tetraacetic acid (EDTA). The developed homogeneous assay can be used in screening molecules that either inhibit or enhance multimerization of sorcin, and the assay format is applicable to various noncompetitive high-throughput screening assays detecting protein multimerization reactions.     

Bidirectional fluorescence resonance energy transfer (FRET) in mutated and chemically modified yellow fluorescent protein (YFP)

Fluorescence resonance energy transfer (FRET) using fluorescent protein variants are used for studying the associations and biomolecular motions of macromolecules inside the cell. Intramolecular FRET utilizing fluorescent chemical labels has been applied in nucleic acid chemistry for detection of specific sequence. However, the biotechnological applications of intramolecular FRET in fluorescent proteins have not been exploited. This study demonstrates the intramolecular FRET between fluorescent protein and conjugated chemical label whereby FRET occurs from inside to outside and vice versa for fluorescent protein. The fluorescent protein is modified for the attachment of chemical fluorophores and the novel FRET pairs created by conjugation are MDCC (435/475)-Citrine (516/529) and Citrine-Alexa fluor (568/603). These protein-label pairs exhibited strong intramolecular FRET and the energy transfer efficiency was determined based on the time evolution of the ratio of emission intensities of labeled and unlabeled proteins. The efficiency was found to be 0.79 and 0.89 for MDCC-Citrine and 0.24 and 0.65 for Citrine-Alexa Fluor pairs when the label is conjugated at different sites in the protein. Fo?rster distance and the average distance between the fluorophores were also determined. The bidirectional approach described here can provide new insights into designing FRET-based sensors.     

Energy-transfer measurements on a double fluorescent labeled ribonuclease A

Two fluorescent groups have been covalently attached to ribonuclease A: first, the alpha-amino group is labeled upon reaction with fluorescein isothiocyanate, and second, one of the active site histidine residues is modified by N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid. Among the products of these two successive chemical modifications, a derivative bearing one label on Lys-1 and the other label on His-119 can be isolated and characterized. Because of their spectral properties, these two fluorophores, fluorescein and N-[(acetamido)ethyl]-5-naphthylamine-1-sulfonic acid, are suitable for measuring resonance energy transfer within a single protein molecule. The efficiency of the energy transfer is close to 100% in the native state and is reduced to about 50% in the guanidine-unfolded state. This efficiency is further diminished upon reduction of the disulfide bonds in denaturing conditions. The efficiency of energy transfer has been determined independently from both emission and excitation spectra of the double-labeled protein, when unfolded with intact disulfide bonds. The average distance between the two fluorescent groups can be obtained from these measurements: it increases from 20 A at most in the native state to 46 A or more in the unfolded state.     

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.     

Homogeneous real-time detection and quantification of nucleic acid amplification using restriction enzyme digestion

A method for real-time fluorescent detection and quantification of nucleic acid amplification using a restriction endonuclease was developed. In this homogeneous system detection is mediated by a primer containing a reporter and quencher moiety at its 5' terminus separated by a short section of DNA encoding a restriction enzyme recognition sequence. In the single stranded form, the signal from the fluorescent reporter is quenched due to fluorescence resonance energy transfer. However, as the primer becomes incorporated into a double stranded amplicon, a restriction enzyme present in the reaction cleaves the DNA linking the reporter and quencher, allowing unrestricted fluorescence of the reporter. To test this system, a primer specific for the E6 gene of human papilloma virus (HPV) 16 was combined with the cleavable energy transfer label and used to amplify HPV16 positive DNA. In the presence of the thermally stable restriction enzyme BstNI, the reporter system was found to generate a fluorescent signal in proportion to the amount of template DNA. In addition to this direct format, the reporter primer was also used to monitor and quantify the amplification of other sequences. This was accomplished by using primers that contain a tag sequence complementary to the reporter oligonucleotide.     

Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells

A conventional fluorescence microscope was modified to observe the sites of resonance energy transfer (RET) between fluorescent probes in model membranes and in living cells. These modifications, and the parameters necessary to observe RET between membrane-bound fluorochromes, are detailed for a system that uses N-4-nitrobenzo-2-oxa-1,3-diazole (NBD) or fluorescein as the energy donor and sulforhodamine as the energy acceptor. The necessary parameters for RET in this system were first optimized using liposomes. Both quenching of the energy donor and sensitized fluorescence of the energy acceptor could be directly observed in the microscope. RET microscopy was then used in cultured fibroblasts to identify those intracellular organelles labeled by the lipid probe, N-SRh-decylamine (N-SRh-C10). This was done by observing the sites of RET in cells doubly labeled with N-SRh-C10 and an NBD-labeled lipid previously shown to label the endoplasmic reticulum, mitochondria, and nuclear envelope. RET microscopy was also used in cells treated with fluorescein-labeled Lens culinaris agglutinin and a sulforhodamine derivative of phosphatidylcholine to examine the internalization of plasma membrane lipid and protein probes. After internalization, the fluorescent lectin resided in most, but not all of the intracellular compartments labeled by the fluorescent lipid, suggesting sorting of the membrane-bound lectin into a subset of internal compartments. We conclude that RET microscopy can co-localize different membrane-bound components at high resolution, and may be particularly useful in examining temporal and spatial changes in the distribution of fluorescent molecules in membranes of the living cell.     

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer (3-FRET). This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.     

Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP

Background  

The green fluorescent protein (GFP) has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors. Mutagenesis of the wildtype gene has yielded a number of improved variants such as EGFP or colour variants suitable for fluorescence resonance energy transfer (FRET). However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins.     

Exploring the structure of the N-terminal domain of CP29 with ultrafast fluorescence spectroscopy

A high-throughput Förster resonance energy transfer (FRET) study was performed on the approximately 100 amino acids long N-terminal domain of the photosynthetic complex CP29 of higher plants. For this purpose, CP29 was singly mutated along its N-terminal domain, replacing one-by-one native amino acids by a cysteine, which was labeled with a BODIPY fluorescent probe, and reconstituted with the natural pigments of CP9, chlorophylls and xanthophylls. Picosecond fluorescence experiments revealed rapid energy transfer (~20–70 ps) from BODIPY at amino-acid positions 4, 22, 33, 40, 56, 65, 74, 90, and 97 to Chl a molecules in the hydrophobic part of the protein. From the energy transfer times, distances were estimated between label and chlorophyll molecules, using the Förster equation. When the label was attached to amino acids 4, 56, and 97, it was found to be located very close to the protein core (~15 Å), whereas labels at positions 15, 22, 33, 40, 65, 74, and 90 were found at somewhat larger distances. It is concluded that the entire N-terminal domain is in close contact with the hydrophobic core and that there is no loop sticking out into the stroma. Most of the results support a recently proposed topological model for the N-terminus of CP29, which was based on electron-spin-resonance measurements on spin-labeled CP29 with and without its natural pigment content. The present results lead to a slight refinement of that model.     

Applications of phasors to in vitro time-resolved fluorescence measurements

The phasor method of treating fluorescence lifetime data provides a facile and convenient approach to characterize lifetime heterogeneity and to detect the presence of excited state reactions such as solvent relaxation and Förster resonance energy transfer. The method uses a plot of M sin(Φ) versus M cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. A principal advantage of the phasor method is that it provides a model-less approach to time-resolved data amenable to visual inspection. Although the phasor approach has been recently applied to fluorescence lifetime imaging microscopy, it has not been used extensively for cuvette studies. In the current study, we explore the applications of the method to in vitro samples. The phasors of binary and ternary mixtures of fluorescent dyes demonstrate the utility of the method for investigating complex mixtures. Data from excited state reactions, such as dipolar relaxation in membrane and protein systems and also energy transfer from the tryptophan residue to the chromophore in enhanced green fluorescent protein, are also presented.     

Genetically encoded FRET-pair on the basis of terbium-binding peptide and red fluorescent protein

The genetically encoded FRET-pair was developed on the basis of terbium-binding peptide and red fluorescent protein DsRed2. To study fluorescence resonance energy transfer within the FRET-pair, the engineered construction was obtained, where sequences of terbium-binding peptide and red fluorescent protein DsRed2 were fused in single reading frame. The expression of this construction in strain E. coli BL21(DE3) was studied and conditions of synthesis, isolation, and purification of recombinant protein were optimized. The hydrodynamic radius of hybrid protein was determined by the method of dynamic diffusion. Energy transfer between sensitized terbium and red fluorescent protein was confirmed by the methods of fluorescence spectroscopy. The obtained FRET-pair may be used both for studies in vitro and as reporters in living cells.     

Time-resolved luminescence energy transfer immunobinding study using a ruthenium-ligand complex as a donor label

A novel immunosystem is described that exploits the effect of luminescence energy transfer from a luminescently labeled antigen to a fluorescent antibody. A luminescent ruthenium-ligand complex (D-455) with absorption/emission maxima at 456/639 nm, respectively, was employed as the donor label, and a squaraine-type cyanine label (636/655 nm), as the fluorescent acceptor label. Specifically, the system human serum albumin (HSA)/anti-HSA was studied. HSA was labeled with the donor dye D-455, and anti-HSA was labeled with the acceptor dye A-631. On formation of the antigen-antibody complex, energy transfer occurs. The radiationless energy transfer affects both the decay time of D-455 and the intensities of the emissions of both D-455 and A-631. The decay time of around 500 ns of D-455 allows frequency-domain measurements in the low kilohertz range and therefore can be based on the use of conventional optoelectronics. This also suggests gated measurements to be performed. The major difference from existing HSA immunosystems is the use of a slow decaying ruthenium-ligand complex as the donor and of a long-wave emitting cyanine acceptor dye having a high quantum yield and a decay kinetics that is governed by the rate of energy transfer from the slow decaying donor.     

Dimerization of corticotropin-releasing factor receptor type 1 is not coupled to ligand binding

As described previously, receptor dimerization of G protein-coupled receptors may influence signaling, trafficking, and regulation in vivo. Up to now, most studies aiming at the possible role of receptor dimerization in receptor activation and signal transduction are focused on class A GPCRs. In the present work, the dimerization behavior of the corticotropin-releasing factor receptor type 1 (CRF1R), which belongs to class B of GPCRs and plays an important role in coordination of the immune response, stress, and learning behavior, was investigated by using fluorescence resonance energy transfer (FRET). For this purpose, we generated fusion proteins of CRF1R tagged at their C-terminus to a cyan or yellow fluorescent protein, which can be used as a FRET pair. Binding studies verified that the receptor constructs were able to bind their natural ligands in a manner comparable with the wild-type receptor, whereas cAMP accumulation proved the functionality of the constructs. In microscopic studies, a dimerization of the CRF1R was observed, but the addition of either CRF-related agonists or antagonists did not show any dose-related increase of the observed FRET signal, indicating that the dimer-monomer ratio is not changed on addition of ligand.     

FRET study of membrane proteins: simulation-based fitting for analysis of membrane protein embedment and association

A new formalism for the simultaneous determination of the membrane embedment and aggregation of membrane proteins is developed. This method is based on steady-state F?rster (or fluorescence) resonance energy transfer (FRET) experiments on site-directed fluorescence labeled proteins in combination with global data analysis utilizing simulation-based fitting. The simulation of FRET was validated by a comparison with a known analytical solution for energy transfer in idealized membrane systems. The applicability of the simulation-based fitting approach was verified on simulated FRET data and then applied to determine the structural properties of the well-known major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants Y24C, G38C, and T46C of this protein were produced and specifically labeled with the fluorescence label AEDANS. The energy transfer data from the natural tryptophan at position 26, which is used as a donor, to AEDANS were analyzed assuming a helix model for the transmembrane domain of the protein. As a result of the FRET data analysis, the topology and bilayer embedment of this domain were quantitatively characterized. The resulting tilt of the transmembrane helix of the protein is 18 +/- 2 degrees. The tryptophan is located at a distance of 8.5 +/- 0.5 A from the membrane center. No specific aggregation of the protein was found. The methodology developed here is not limited to M13 major coat protein and can be used in principle to study the bilayer embedment of any small protein with a single transmembrane domain.     

Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET

Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent, noncanonical amino acid, L-ANAP. A metal chelating lipid was used to decorate the plasma membrane with high-density/high-affinity metal-binding sites. The fluorescence resonance energy transfer (FRET) efficiencies between L-ANAP in TRPV1 and Co²⁺ bound to the plasma membrane were consistent with the arrangement of the ARDs in recent cryoelectron microscopy structures of TRPV1. No change in tmFRET was observed with the TRPV1 agonist capsaicin. These results demonstrate the power of tmFRET for measuring structure and rearrangements of membrane proteins relative to the cell membrane.     

In vivo interaction between dynamitin and MacMARCKS detected by the fluorescent resonance energy transfer method

Dynamitin is a subunit of the dynactin complex regulating microtubule-dependent motor functions, and MacMARCKS (Macrophage-enriched myristoylated alanine-rich protein kinase C substrate) is a major protein kinase C substrate regulating integrin activation. The interaction between dynamitin and MacMARCKS has been implicated in integrin-dependent cell spreading. However, the in vivo interaction of these two proteins in living cells has not been demonstrated. Spatial and temporal information about the interaction is also lacking. In this study, we used the fluorescent resonance energy transfer method to demonstrate in vivo interaction between MacMARCKS and dynamitin with cyan fluorescent protein (CFP)-conjugated dynamitin as the donor fluorophore and yellow fluorescent protein (YFP)-conjugated MacMARCKS as the acceptor fluorophore. The interaction of these two fusion proteins was studied both in vitro and in vivo, and typical fluorescent resonance energy transfer was observed; the CFP emission peak increased while the YFP emission peak decreased when protein interaction was abolished. Spatial and temporal information was obtained in RAW macrophage cells. In resting macrophage cells, dynamitin-MacMARCKS interaction is concentrated at the cell periphery, although the majority of dynamitin is distributed at the perinuclear region of the cells. When cells were treated with phorbol 12-myristate 13-acetate, both proteins concentrated to perinuclear regions of the cells, and yet the interaction disappeared as the cell spread. Similar events were also observed in 293 cells. Thus, we conclude that dynamitin and MacMARCKS indeed interact in living cells.     

Physical association between the adipocyte fatty acid-binding protein and hormone-sensitive lipase: a fluorescence resonance energy transfer analysis

Previous in vitro studies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 microm forskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet.     

A genetically encoded Förster resonance energy transfer biosensor for two-photon excitation microscopy

Pippi (phosphatidyl inositol phosphate indicator) is a biosensor based on the principle of FRET (F?rster resonance energy transfer), which consists of a pair of fluorescent proteins, CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein), the PH domain sandwiched between them, and K-Ras C-terminal sequence for plasma membrane localization. Due to marked cross-excitation of YFP with the conditions used to excite CFP, initial FRET images obtained by TPE (two-photon excitation) microscopy suffered from low signal-to-noise ratio, hampering the observation of lipids in three-dimensional structures. To solve this problem, YFP and CFP in the original Pippi-PI(3,4)P(2) was replaced by sREACh (super resonance energy accepting chromoprotein) and mTFP1 (monomeric teal fluorescent protein), respectively. The biosensor was also fused with an internal control protein, mKeima, where Keima/mTFP1 indicates the FRET efficiency, and indeed epidermal growth factor stimulation increased Keima/mTFP1 in HeLa cells. This biosensor successfully showed PI(3,4)P(2) accumulation to the lateral membrane in the MDCK cyst cultured in a three-dimensional environment. Furthermore, other FRET-based biosensors for PIP(3) distribution and for tyrosine kinase activity were developed based on this method, suggesting its broad application for visualizing signal transduction events with TPE microscopy.