A new pair for inter- and intra-molecular FRET measurement

Fluorescence resonance energy transfer between mutant green fluorescent proteins provides powerful means to monitor in vivo protein-protein proximity and intracellular signaling. However, the current widely applied FRET pair of this class (CFP/YFP) requires excitation by expensive UV lasers, thereby hindering FRET imaging on many confocal microscopes. Further challenges arise from the large spectral overlap of CFP/YFP emission. Another FRET pair GFP/DsRed could obviate such limitations. However, the use of DsRed as a FRET acceptor is hampered by several critical problems, including a slow and incomplete maturation and obligate tetramerization. A tandem dimer mutant of DsRed (TDimer2) has similar spectral properties as those of DsRed. The rapid maturation and non-oligomerization make TDimer2 a promising substitute for DsRed in FRET experiments. Here, we have explored the possibility of using TDimer2 as a FRET acceptor for the donor EGFP. FRET was demonstrated between the EGFP-TDimer2 chimeric fusion protein. By substituting CFP/YFP in the Ca2+-sensor cameleon with EGFP/TDimer2, dynamic changes in cytosolic free Ca2+ concentrations were observed with 488nm excitation under conventional wide-field microscopy. The EGFP/TDimer2 pair was further successfully employed to monitor inter-molecular interaction between Syntaxin and SNAP25. These results reveal EGFP/TDimer2 as a promising FRET pair in monitoring intra-molecular conformation change as well as inter-molecular interaction.     

Quantification of protein interaction in living cells by two-photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed-through

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells.     

Imaging FRET between spectrally similar GFP molecules in single cells

Fluorescence resonance energy transfer (FRET) detection in fusion constructs consisting of green fluorescent protein (GFP) variants linked by a sequence that changes conformation upon modification by enzymes or binding of ligands has enabled detection of physiological processes such as Ca(2+) ion release, and protease and kinase activity. Current FRET microscopy techniques are limited to the use of spectrally distinct GFPs such as blue or cyan donors in combination with green or yellow acceptors. The blue or cyan GFPs have the disadvantages of less brightness and of autofluorescence. Here a FRET imaging method is presented that circumvents the need for spectral separation of the GFPs by determination of the fluorescence lifetime of the combined donor/acceptor emission by fluorescence lifetime imaging microscopy (FLIM). This technique gives a sensitive, reproducible, and intrinsically calibrated FRET measurement that can be used with the spectrally similar and bright yellow and green fluorescent proteins (EYFP/EGFP), a pair previously unusable for FRET applications. We demonstrate the benefits of this approach in the analysis of single-cell signaling by monitoring caspase activity in individual cells during apoptosis.     

An improved cyan fluorescent protein variant useful for FRET

Many genetically encoded biosensors use F?rster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.     

Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins.

BACKGROUND: The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. METHODS: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. RESULTS: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. CONCLUSIONS: We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.     

单个活细胞中生物分子动态行为的FRET实时检测

分别采用两种不同绿色荧光蛋白(green fluorescent prote in,GFP)突变体作为荧光共振能量转移(fluo-rescence resonance energy transfer,FRET)对的供体和受体,并利用分子生物学技术将供体和受体分子分别与特定的生物分子融合,这种技术已经成为在单个活细胞中实时长时间检测蛋白质间的动态相互作用的主要技术。主要介绍了基于GFPs的FRET技术在单个活细胞中实时长时间研究生物分子动态行为的应用。     

Resonance energy transfer between green fluorescent protein variants: complexities revealed with myosin fusion proteins

Green fluorescent protein and its variants are frequently used as F?rster (fluorescence) resonance energy transfer (FRET) pairs to determine the proximity of protein domains. We prepared fusion proteins comprising yellow fluorescent protein-Dictyostelium myosin II motor domain-cyan fluorescent protein (YFP-myosin-CFP) and compared their FRET properties with an existing construct (GFP-myosin-BFP), containing a green fluorescent protein acceptor and blue fluorescent protein donor [Suzuki, Y., Yasunaga, T., Ohkura, R., Wakabayashi, T. and Sutoh, K. (1998) Nature 396, 380-383]. The latter construct showed an apparent 40% reduction in acceptor fluorescence on ATP addition, when excited via the donor, compared with the YFP-myosin-CFP constructs which showed a small increase (相似文献   

Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements

Fluorescence Resonance Energy Transfer (FRET) using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM).     

Antisense activity detection by inhibition of fluorescence resonance energy transfer.

Use of antisense nucleic acids to modulate expression of particular genes is a promising approach to the therapy of human papillomavirus type 16 (HPV-16)-associated cervical cancer. Understandably, evaluation of the in vivo performance of synthetic antisense oligodeoxynucleotides (AS-ODNs) or ribozymes is of ultimate importance to development of effective antisense tools. Here we report the use of a bacterial reporter system based on the inhibition of fluorescence resonance energy transfer (FRET) to measure the interaction of AS-ODNs with HPV-16 target nt 410-445, using variants of the green fluorescent protein (GFP). An optimal FRET-producing pair was selected with GFP as the donor and yellow fluorescent protein (YFP) as the acceptor molecule. Hybridization of AS-ODNs with a chimaeric mRNA containing the antisense target site flanked by GFP variants resulted in the inhibition of the FRET effect. Use of different linkers suggested that the amino acid content of the linker has no significant effect on FRET effect. Antisense accessibility, tested by RNaseH assays with phosphorothioated target-specific and mutant AS-ODNs, suggested a specific effect on the chimaeric mRNA. FRET inhibition measurements correlated with the presence of truncated proteins confirming true antisense activity over the target. Therefore, FRET inhibition may be used for the direct measurement of AS-ODNs activity in vivo.     

APPL Proteins FRET at the BAR: Direct Observation of APPL1 and APPL2 BAR Domain-Mediated Interactions on Cell Membranes Using FRET Microscopy

Background

Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.

Methodology

Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.

Conclusions

All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies.     

Cerulean, Venus, and VenusY67C FRET reference standards

F?rster's resonance energy transfer (FRET) can be used to study protein-protein interactions in living cells. Numerous methods to measure FRET have been devised and implemented; however, the accuracy of these methods is unknown, which makes interpretation of FRET efficiency values difficult if not impossible. This problem exists due to the lack of standards with known FRET efficiencies that can be used to validate FRET measurements. The advent of spectral variants of green fluorescent protein and easy access to cell transfection technology suggests a simple solution to this problem: the development of genetic constructs with known FRET efficiencies that can be replicated with high fidelity and freely distributed. In this study, fluorescent protein constructs with progressively larger separation distances between donors and acceptors were generated and FRET efficiencies were measured using fluorescence lifetime spectroscopy, sensitized acceptor emission, and spectral imaging. Since the results from each method were in good agreement, the FRET efficiency value of each construct could be determined with high accuracy and precision, thereby justifying their use as standards.     

Combination of novel green fluorescent protein mutant TSapphire and DsRed variant mOrange to set up a versatile in planta FRET-FLIM assay

Förster resonance energy transfer (FRET) measurements based on fluorescence lifetime imaging microscopy (FLIM) are increasingly being used to assess molecular conformations and associations in living systems. Reduction in the excited-state lifetime of the donor fluorophore in the presence of an appropriately positioned acceptor is taken as strong evidence of FRET. Traditionally, cyan fluorescent protein has been widely used as a donor fluorophore in FRET experiments. However, given its photolabile nature, low quantum yield, and multiexponential lifetime, cyan fluorescent protein is far from an ideal donor in FRET imaging. Here, we report the application and use of the TSapphire mutant of green fluorescent protein as an efficient donor to mOrange in FLIM-based FRET imaging in intact plant cells. Using time-correlated single photon counting-FLIM, we show that TSapphire expressed in living plant cells decays with lifetime of 2.93 ± 0.09 ns. Chimerically linked TSapphire and mOrange (with 16-amino acid linker in between) exhibit substantial energy transfer based on the reduction in the lifetime of TSapphire in the presence of the acceptor mOrange. Experiments performed with various genetically and/or biochemically known interacting plant proteins demonstrate the versatility of the FRET-FLIM system presented here in different subcellular compartments tested (cytosol, nucleus, and at plasma membrane). The better spectral overlap with red monomers, higher photostability, and monoexponential lifetime of TSapphire makes it an ideal FRET-FLIM donor to study protein-protein interactions in diverse eukaryotic systems overcoming, in particular, many technical challenges encountered (like autofluorescence of cell walls and fluorescence of pigments associated with photosynthetic apparatus) while studying plant protein dynamics and interactions.Single- and dual-color fluorescence imaging with intrinsically fluorescent proteins is increasingly being used to study the expression, targeting, colocalization, turnover, and associations of diverse proteins involved in different plant signal transduction pathways (for review, see Fricker et al., 2006). Concurrent with the use of fluorescence-based cell biology, Förster resonance energy transfer (FRET) has emerged as a convenient tool to study the dynamics of protein associations in vivo. The technique exploits the biophysical phenomenon of nonradiative energy transfer from a donor fluorophore to an appropriately positioned acceptor at a nanometer scale (1–10 nm; Jares-Erijman and Jovin, 2003). In living cells, FRET occurs when two proteins (or different domains within a single protein) fused to suitable donor and acceptor fluorophores physically interact, thus bringing the donor and the acceptor within the favorable proximity for energy transfer (Immink et al., 2002; Bhat et al., 2006). This results in a decrease in the donor''s fluorescence intensity (or quantum yield [QY]) and excited-state lifetime (Gadella et al., 1999). Furthermore, if the acceptor molecule is a fluorophore, then FRET additionally results in an increase in the acceptor''s emission intensity (sensitized emission; Shah et al., 2001; Bhat et al., 2006).However, the exploitation and use of fluorescent marker proteins to study protein trafficking and associations in plants can be problematic because plant cells contain a number of autofluorescent compounds (e.g. lignin, chlorophyll, phenols, etc.) whose emission spectra interfere with that of the most commonly used green or red fluorescent protein fluorophores and/or their spectral variants. For example, lignin fluorescence in roots, vascular tissues, and cell walls of aerial plant parts interferes with imaging at wavelengths between 490 and 620 nm, whereas the chlorophyll autofluorescence in green aerial plant parts is prevalent between 630 and 770 nm (Chapman et al., 2005). Consequently, conventional imaging of GFP and its closest spectral variants (like cyan fluorescent protein [CFP] and yellow fluorescent protein [YFP]) is most likely to be problematic in roots, whereas red-shifted intrinsic fluorescent proteins (including monomeric red fluorescent protein and recently identified spectral variants like mStrawberry and mCherry) may be hard to discriminate in chloroplast-containing aerial tissues (Chapman et al., 2005). The problems get further compounded in FRET assays because the autofluorescence arising from phenols, lignin, and chlorophyll can limit the choice of fluorophores suitable for in planta FRET assays.CFP and YFP have been widely used as a donor-acceptor pair in in planta FRET measurements (Bhat et al., 2006; Dixit et al., 2006). However, in photophysical terms, this pair is less than ideal for FRET imaging. Both have broad excitation and emission spectra with a small Stokes shift (Chapman et al., 2005). Second, QY of CFP (QY = 0.4) is relatively lower than that of YFP (QY = 0.61), and thus a significantly higher (and rather cell damaging) amount of excitation energy is needed to induce FRET (Dixit et al., 2006). Additionally, CFP displays multiexponential lifetimes with a shorter (1.3 ns) and a longer (2.6 ns) component (Becker et al., 2006). Although the deviation from the single-component decay is reasonably small (Tramier et al., 2002; Becker et al., 2006), the shorter CFP lifetime component can erroneously be interpreted as being the result of lifetime reduction due to energy transfer. At the same time, weak or transient protein associations may get masked and thus remain undetected. Whereas the parental wild-type GFP is extremely photostable and shows a monoexponential decay pattern (excited-state lifetime 3.16 ± 0.03 ns; Striker et al., 1999; Volkmer et al., 2000; Shaner et al., 2005), its close spectral overlap with YFP makes it unsuitable as a donor in GFP-YFP FRET experiments. Likewise, wild-type or enhanced GFP (or YFP) as a donor to red-shifted monomers as acceptors is suboptimal because the 488-nm (or 514-nm) laser line commonly used to excite GFP (or YFP) cross excites most of the red monomers (e.g. mOrange, mStrawberry) because of their broad excitation spectra (Zapata-Hommer and Griesbeck, 2003; Shaner et al., 2004).Recently, TSapphire (Q69M/C70P/V163A/S175G; excitation/emission 399/511 nm), a variant of the Sapphire (T203I) mutant of wild-type GFP with improved folding properties and better pH sensitivity, was described (Zapata-Hommer and Griesbeck, 2003). The T203I mutation in TSapphire (and original Sapphire as well) abolishes the 475-nm excitation peak found in the wild-type GFP (Tsien, 1998). TSapphire is efficiently excited below 410 nm, which makes it ideal for studying plant protein dynamics and interactions because, at this wavelength, there is negligible excitation of the autofluorescing chlorophyll pigments. Furthermore, TSapphire also represents a good donor to red monomer acceptors that are negligibly excited at this wavelength (Shaner et al., 2004). Using a purified Zn²⁺ sensor with TSapphire and mOrange as a donor-acceptor pair, Shaner and colleagues demonstrated the ratiometric intramolecular FRET between the two fluorophores in vitro (Shaner et al., 2004). The sensor yielded a 6-fold ratiometric increase (562/514-nm mOrange/TSapphire emission ratio) upon Zn²⁺ binding.However, currently there are no reports demonstrating the application and use of TSapphire and monomeric red-shifted fluorophores as donor-acceptor FRET pairs to probe intermolecular protein-protein interactions in vivo. In this article, we demonstrate in vivo FRET-fluorescence lifetime imaging microscopy (FLIM) between the donor TSapphire and the acceptor mOrange. We show that TSapphire expressed in living plant cells decays with a monoexponential lifetime of 2.93 ± 0.09 ns, which is in agreement with the published lifetime for its parent wild-type GFP (3.2 ns; Striker et al., 1999; Volkmer et al., 2000). Furthermore, we demonstrate intramolecular FRET-FLIM between chimerically linked TSapphire and mOrange (with a 16-amino acid linker in between). When fused to genetically known interacting proteins and expressed in intact living cells, the donor and the acceptor fluorophores show energy transfer in different subcellular compartments indicative of intermolecular protein-protein interactions. These results validate the versatility of the proposed in vivo FRET-FLIM assay based on the donor TSapphire and the acceptor mOrange, which turns out to work with both soluble and membrane proteins.     

FRET between cardiac Na+ channel subunits measured with a confocal microscope and a streak camera

When and where proteins associate is a central question in many biomolecular studies. F?rster resonance energy transfer (FRET) measurements can be used to address this question when the interacting proteins are labeled with appropriate donor and acceptor fluorophores. We describe an improved method to determine FRET efficiency that uses a mode-locked laser, a confocal microscope and a streak camera. We applied this method to study the association of alpha and beta(1) subunits of the human cardiac sodium channel. The subunits were tagged with the cyan and yellow variants of the green fluorescent protein (GFP) and expressed in human embryonic kidney (HEK293) cells. Pronounced FRET between the channel subunits in the endoplasmic reticulum (ER) suggested that the subunits associate before they reach the plasma membrane. The described method allows simultaneous measurement of donor and acceptor fluorescence decays and provides an intrinsically validated estimate of FRET efficiency.     

Quantitative FRET measurement based on spectral unmixing of donor,acceptor and spontaneous excitation‐emission spectra

The spontaneous excitation‐emission (ExEm) spectrum is introduced to the quantitative mExEm‐spFRET methodology we recently developed as a spectral unmixing component for quantitative fluorescence resonance energy transfer measurement, named as SPEES‐FRET method. The spectral fingerprints of both donor and acceptor were measured in HepG2 cells with low autofluorescence separately expressing donor and acceptor, and the spontaneous spectral fingerprint of HEK293 cells with strong autofluoresence was measured from blank cells. SPEES‐FRET was performed on improved spectrometer‐microscope system to measure the FRET efficiency (E) and concentration ratio (R _C) of acceptor to donor vales of FRET tandem plasmids in HEK293 cells, and obtained stable and consistent results with the expected values. Moreover, SPEES‐FRET always obtained stable results for the bright and dim cells coexpressing Cerulean and Venus or Cyan Fluorescent Protein (CFP)‐Bax and Yellow fluorescent protein (YFP)‐Bax, and the E values between CFP‐Bax and YFP‐Bax were 0.02 for healthy cells and 0.14 for the staurosporine (STS)‐treated apoptotic cells. Collectively, SPEES‐FRET has very strong robustness against cellular autofluorescence, and thus is applicable to quantitative evaluation on the protein‐protein interaction in living cells with strong autofluoresence.      

Protein localization in living cells and tissues using FRET and FLIM

Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution. One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength. Mathematical algorithms are required to correct the spectral bleed-through signal in wide-field, confocal, and two-photon FRET microscopy. In contrast, spectral bleed-through is not an issue in FRET/FLIM imaging because only the donor fluorophore lifetime is measured; also, fluorescence lifetime imaging microscopy (FLIM) measurements are independent of excitation intensity or fluorophore concentration. The combination of FRET and FLIM provides high spatial (nanometer) and temporal (nanosecond) resolution when compared to intensity-based FRET imaging. In this paper, we describe various FRET microscopy techniques and its application to protein-protein interactions.     

Detection of oligomerization and conformational changes in the Na+/H+ antiporter from Helicobacter pylori by fluorescence resonance energy transfer

Oligomerization and conformational changes in the Na+/H+ antiporter from Helicobacter pylori (HPNhaA) were studied by means of fluorescence resonance energy transfer (FRET) analysis. Na+/H+ antiporter-deficient Escherichia coli cells expressing C-terminal fusions of HPNhaA to green fluorescent protein (GFP) variants exhibited wild-type levels of antiporter activity in their everted membrane vesicles. Vesicles containing both HPNhaA-CFP and HPNhaA-YFP or HPNhaA-Venus exhibited FRET from CFP (donor) to YFP or Venus (acceptor), suggesting that HPNhaA forms an oligomer. Co-precipitation of HPNhaA tagged by Venus and FLAG sequences confirmed oligomerization. FRET decreased extensively after treatment of the vesicles with proteinase K, which released GFP variants from the fusion proteins. FRET was not observed by merely mixing vesicles expressing the donor or acceptor fusion alone. Fluorescence of Venus is less sensitive to anions and stronger than that of anion-sensitive YFP. Using HPNhaA-Venus as the acceptor, Li+ was found to cause a significant decrease in FRET regardless of the presence or absence of DeltapH across the membranes, whereas Na+ caused a much weaker effect. This Li+ effect was minimal in vesicles prepared from cells expressing HPNhaA containing an Asp141 to Asn mutation, which results in defective Li+/H+ antiporter activity, possibly Li+ binding. These results demonstrate that monomer interactions within the HPNhaA oligomer are weakened possibly by Li+ binding. Dynamic interactions between HPNhaA monomers were detectable in membranes by FRET analysis, thus providing a new approach to study dynamic conformational changes in NhaA during antiport activity.     

Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein

BACKGROUND: Fluorescence resonance energy transfer (FRET) is a powerful technique for measuring molecular interactions at Angstrom distances. We present a new method for FRET that utilizes the unique spectral properties of variants of the green fluorescent protein (GFP) for large-scale analysis by flow cytometry. METHODS: The proteins of interest are fused in frame separately to the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP). FRET between these differentially tagged fusion proteins is analyzed using a dual-laser FACSVantage cytometer. RESULTS: We show that homotypic interactions between individual receptor chains of tumor necrosis factor receptor (TNFR) family members can be detected as FRET from CFP-tagged receptor chains to YFP-tagged receptor chains. Noncovalent molecular complexation can be detected as FRET between fusions of CFP and YFP to either the intracellular or extracellular regions of the receptor chains. The specificity of the assay is demonstrated by the absence of FRET between heterologous receptor pairs that do not biochemically associate with each other. Interaction between a TNFR-like receptor (Fas/CD95/Apo-1) and a downstream cytoplasmic signaling component (FADD) can also be demonstrated by flow cytometric FRET analysis. CONCLUSIONS: The utility of spectral variants of GFP in flow cytometric FRET analysis of membrane receptors is demonstrated. This method of analyzing FRET allows probing of noncovalent molecular interactions that involve both the intracellular and extracellular regions of membrane proteins as well as proteins within the cells. Unlike biochemical methods, FRET allows the quantitative determination of noncovalent molecular associations at Angstrom level in living cells. Moreover, flow cytometry allows quantitative analyses to be carried out on a cell-by-cell basis on large number of cells. Published 2001 Wiley-Liss, Inc.     

A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.     

The Impact of Heterogeneity and Dark Acceptor States on FRET: Implications for Using Fluorescent Protein Donors and Acceptors

Förster resonance energy transfer (FRET) microscopy is widely used to study protein interactions in living cells. Typically, spectral variants of the Green Fluorescent Protein (FPs) are incorporated into proteins expressed in cells, and FRET between donor and acceptor FPs is assayed. As appreciable FRET occurs only when donors and acceptors are within 10 nm of each other, the presence of FRET can be indicative of aggregation that may denote association of interacting species. By monitoring the excited-state (fluorescence) decay of the donor in the presence and absence of acceptors, dual-component decay analysis has been used to reveal the fraction of donors that are FRET positive (i.e., in aggregates)._However, control experiments using constructs containing both a donor and an acceptor FP on the same protein repeatedly indicate that a large fraction of these donors are FRET negative, thus rendering the interpretation of dual-component analysis for aggregates between separately donor-containing and acceptor-containing proteins problematic. Using Monte-Carlo simulations and analytical expressions, two possible sources for such anomalous behavior are explored: 1) conformational heterogeneity of the proteins, such that variations in the distance separating donor and acceptor FPs and/or their relative orientations persist on time-scales long in comparison with the excited-state lifetime, and 2) FP dark states.     

Improving the flexibility of genetically encoded voltage indicators via intermolecular FRET

A new family of genetically encoded voltage indicators (GEVIs) has been developed based on intermolecular Förster resonance energy transfer (FRET). To test the hypothesis that the GEVI ArcLight functions via interactions between the fluorescent protein (FP) domains of neighboring probes, the FP of ArcLight was replaced with either a FRET donor or acceptor FP. We discovered relatively large FRET signals only when cells were cotransfected with both the FRET donor and acceptor GEVIs. Using a cyan fluorescent protein donor and an RFP acceptor, we were able to observe a voltage-dependent signal with an emission peak separated by over 200 nm from the excitation wavelength. The intermolecular FRET strategy also works for rhodopsin-based probes, potentially improving their flexibility as well. Separating the FRET pair into two distinct proteins has important advantages over intramolecular FRET constructs. The signals are larger because the voltage-induced conformational change moves two FPs independently. The expression of the FRET donor and acceptor can also be restricted independently, enabling greater cell type specificity as well as refined subcellular voltage reporting.