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.     

High-precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion protein

We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donor-acceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements.     

Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius

Enhanced cyan and yellow fluorescent proteins are widely used for dual color imaging and protein-protein interaction studies based on fluorescence resonance energy transfer. Use of these fluorescent proteins can be limited by their thermosensitivity, dim fluorescence, and tendency for aggregation. Here we report the results of a site-directed mutagenesis approach to improve these fluorescent proteins. We created monomeric optimized variants of ECFP and EYFP, which fold faster and more efficiently at 37 degrees C and have superior solubility and brightness. Bacteria expressing SCFP3A were 9-fold brighter than those expressing ECFP and 1.2-fold brighter than bacteria expressing Cerulean. SCFP3A has an increased quantum yield (0.56) and fluorescence lifetime. Bacteria expressing SYFP2 were 12 times brighter than those expressing EYFP(Q69K) and almost 2-fold brighter than bacteria expressing Venus. In HeLa cells, the improvements were less pronounced; nonetheless, cells expressing SCFP3A and SYFP2 were both 1.5-fold brighter than cells expressing ECFP and EYFP(Q69K), respectively. The enhancements of SCFP3A and SYFP2 are most probably due to an increased intrinsic brightness (1.7-fold and 1.3-fold for purified recombinant proteins, compared to ECFP & EYFP(Q69K), respectively) and due to enhanced protein folding and maturation. The latter enhancements most significantly contribute to the increased fluorescent yield in bacteria whereas they appear less significant for mammalian cell systems. SCFP3A and SYFP2 make a superior donor-acceptor pair for fluorescence resonance energy transfer, because of the high quantum yield and increased lifetime of SCFP3A and the high extinction coefficient of SYFP2. Furthermore, SCFP1, a CFP variant with a short fluorescence lifetime but identical spectra compared to ECFP and SCFP3A, was characterized. Using the large lifetime difference between SCFP1 and SCFP3A enabled us to perform for the first time dual-lifetime imaging of spectrally identical fluorescent species in living cells.     

High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy

Detection of Forster resonance energy transfer (FRET) between fluorescent protein labeled targets is a valuable strategy for measurement of protein-protein interactions and other intracellular processes. Despite the utility of FRET, widespread application of this technique to biological problems and high-throughput screening has been limited by low-contrast measurement strategies that rely on the detection of sensitized emission or photodestruction of the sample. Here we report a FRET detection strategy based on detecting depolarized sensitized emission. In the absence of FRET, we show that fluorescence emission from a donor fluorescent protein is highly polarized. Depolarization of fluorescence emission is observed only in the presence of energy transfer. A simple detection strategy was adapted for fluorescence microscopy using both laser scanning and wide-field approaches. This approach is able to distinguish FRET between linked and unlinked Cerulean and Venus fluorescent proteins in living cells with a larger dynamic range than other approaches.     

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).     

ATP changes the fluorescence lifetime of cyan fluorescent protein via an interaction with His148

Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions.     

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive.     

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.     

Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation

Background

The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.

Methodology/Principal Findings

When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.

Conclusions/Significance

Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions.     

Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging

The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.     

Fluorescent proteins for single‐molecule fluorescence applications

We present single‐molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single‐molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single‐molecule applications, demonstrated by high photostability and rare fluorescence‐intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single‐molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions

We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET using multiphoton fluorescence lifetime imaging microscopy (FLIM). The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) alpha in carcinoma cells for both live- and fixed-cell experiments. The CXCR4-EGFP: PKCalpha-mRFP1 complex was found to be localized precisely to intracellular vesicles and cell protrusions when imaged by multiphoton fluorescence-FLIM. A comparison of the FRET efficiencies obtained using mRFP1-tagged regulatory domain or full-length PKCalpha as the acceptor revealed that PKCalpha, in the closed (inactive) form, is restrained from associating with the cytoplasmic portion of CXCR4. Live-cell FLIM experiments show that the assembly of this receptor:kinase complex is concomitant with the endocytosis process. This is confirmed by experimental evidence suggesting that the recycling of the CXCR4 receptor is increased on stimulation with phorbol ester and blocked on inhibition of PKC by bisindolylmaleimide. The EGFP-mRFP1 couple should be widely applicable, particularly to live-cell quantitative FRET assays.     

X-ray structure of Cerulean GFP: a tryptophan-based chromophore useful for fluorescence lifetime imaging

The crystal structure of the cyan-fluorescent Cerulean green fluorescent protein (GFP), a variant of enhanced cyan fluorescent protein (ECFP), has been determined to 2.0 A. Cerulean bears an internal fluorophore composed of an indole moiety derived from Y66W, conjugated to the GFP-like imidazolinone ring via a methylene bridge. Cerulean undergoes highly efficient fluorescence resonance energy transfer (FRET) to yellow acceptor molecules and exhibits significantly reduced excited-state heterogeneity. This feature was rationally engineered in ECFP by substituting His148 with an aspartic acid [Rizzo et al. (2004) Nat. Biotechnol. 22, 445], rendering Cerulean useful for fluorescence lifetime imaging microscopy (FLIM). The X-ray structure is consistent with a single conformation of the chromophore and surrounding residues and may therefore provide a structural rationale for the previously described monoexponential fluorescence decay. Unexpectedly, the carboxyl group of H148D is found in a buried position, directly contacting the indole nitrogen of the chromophore via a bifurcated hydrogen bond. Compared to the similarly constructed ECFP chromophore, the indole group of Cerulean is rotated around the methylene bridge to adopt a cis-coplanar conformation with respect to the imidazolinone ring, resulting in a close edge-to-edge contact of the two ring systems. The double-humped absorbance spectrum persists in single-crystal absorbance measurements, casting doubt on the idea that ground state conformational heterogeneity forms the basis of the two overlapping transitions. At low pH, a blue shift in absorbance of 10-15 nm suggests a pH-induced structural transition that proceeds with a time constant of 47 (+/-2) min and is reversible. Possible interpretations in terms of chromophore isomerization are presented.     

Visualization of ternary complexes in living cells by using a BiFC-based FRET assay

Studies of protein interactions have increased our understanding and knowledge of biological processes. Assays that utilize fluorescent proteins, such as fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), have enabled direct visualization of protein interactions in living cells. However, these assays are primarily suitable for a pair of interacting proteins, and methods to visualize and identify multiple protein complexes in vivo are very limited. This protocol describes the recently developed BiFC-FRET assay, which allows visualization of ternary complexes in living cells. We discuss how to design the BiFC-FRET assay on the basis of the validation of BiFC and FRET assays and how to perform transfection experiments for acquisition of fluorescent images for net FRET calculation. We also provide three methods for normalization of the FRET efficiency. The assay employs a two-chromophore and three-filter FRET setup and is applicable to epifluorescence microscopes. The entire protocol takes about 2-3 weeks to complete.     

Biophysical characterization of natural and mutant fluorescent proteins cloned from zooxanthellate corals

Two novel colored fluorescent proteins were cloned and biophysically characterized from zooxanthellate corals (Anthozoa). A cyan fluorescent protein derived from the coral Montastrea cavernosa (mcCFP) is a trimeric complex with strong blue-shifted excitation and emission maxima at 432 and 477 nm, respectively. The native complex has a fluorescence lifetime of 2.66 ± 0.01 ns and an inferred absolute quantum yield of 0.385. The spectroscopic properties of a green fluorescent protein cloned from Meandrina meandrites (mmGFP) resemble the commercially available GFP derived originally from the hydrozoan Aequorea victoria (avGFP). mmGFP is a monomeric protein with an excitation maximum at 398 nm and an emission maximum at 505 nm, a fluorescence lifetime of 3.10 ± 0.01 ns and an absolute quantum yield of 0.645. Sequence homology with avGFP and the red fluorescent protein (DsRed) indicates that the proteins adopt the classic β-barrel configuration with 11 β-strands. The three amino acid residues that comprise the chromophore are QYG for mcCFP and TYG for mmGFP, compared with SYG for avGFP. A single point mutation, Ser-110 to Asn, was introduced into mmGFP by random mutagenesis. Denaturation and refolding experiments showed that the mutant has reduced aggregation, increased solubility and more efficient refolding relative to the wild type. Time-resolved emission lifetimes and anisotropies suggest that the electronic structure of the chromophore is highly dependent on the protonation state of adjoining residues.     

Quantitative imaging of protein interactions in the cell nucleus

Over the past decade, genetically encoded fluorescent proteins have become widely used as noninvasive markers in living cells. The development of fluorescent proteins, coupled with advances in digital imaging, has led to the rapid evolution of live-cell imaging methods. These approaches are being applied to address biological questions of the recruitment, co-localization, and interactions of specific proteins within particular subcellular compartments. In the wake of this rapid progress, however, come important issues associated with the acquisition and analysis of ever larger and more complex digital imaging data sets. Using protein localization in the mammalian cell nucleus as an example, we will review some recent developments in the application of quantitative imaging to analyze subcellular distribution and co-localization of proteins in populations of living cells. In this report, we review the principles of acquiring fluorescence resonance energy transfer (FRET) microscopy measurements to define the spatial relationships between proteins. We then discuss how fluorescence lifetime imaging microscopy (FLIM) provides a method that is independent of intensity-based measurements to detect localized protein interactions with spatial resolution. Finally, we consider potential problems associated with the expression of proteins fused to fluorescent proteins for FRET-based measurements from living cells.     

Expression, purification, crystallization and preliminary x-ray analysis of Cyan fluorescent protein CyPet

The technique of fluorescence (or F?rster) resonance energy transfer (FRET) is widely used to observe bimolecular interaction in living cells. Cyan and yellow fluorescent proteins are the most widely used pair in FRET analysis. CyPet and YPet are two newly optimized fluorescent proteins that have much better dynamic range and sensitivity than CFP/YFP pair, although the crystallographic structure and the mechanism of better fluorescent characteristics of CyPet are still unknown. We have expressed the cyan fluorescent protein CyPet using pT7 prokaryocyte expression system in Escherichia coli strain Rosetta (DE3) pLysS by auto-induction. After purification, the recombinant CyPet protein was crystallized by hanging drop vapor diffusion technique and could diffract to 2.55A resolution. The data showed that the orthorhombic CyPet crystal was in space group P212121 with unit cell parameters (51.55, 61.53, 63.36) and contained one molecule in one asymmetric unit.     

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.     

Live cell detection of caspase-3 activation by a Discosoma-red-fluorescent-protein-based fluorescence resonance energy transfer construct

A probe consisting of Discosoma red fluorescent protein (DsRed) and enhanced yellow fluorescent protein (EYFP) linked by a 19-amino-acid chain containing the caspase-3 cleavage site Asp-Glu-Val-Asp was developed to monitor caspase-3 activation in living cells. The expression of the tandem construct in mammalian cells yielded a strong red fluorescence when excited with 450- to 490-nm light or with a 488-nm argon ion laser line as a result of fluorescence resonance energy transfer (FRET) from donor EYFP to acceptor DsRed. The advantage over previous constructs using cyan fluorescent protein is that our construct can be used when excitation wavelengths lower than 488nm are not available. To validate the construct, murine HT-22 hippocampal neuronal cells were triggered to undergo CD95-induced neuronal death. An increase in caspase-3 activity was demonstrated by a reduction of FRET in cells transfected with the construct. This was manifested by a dequenching of EYFP fluorescence leading to an increase in EYFP emission and a corresponding decrease in DsRed fluorescence, which correlated with an increase in pro-caspase-3 processing. We conclude that CD95-induced caspase-3 activation in HT-22 cells was readily detected at the single-cell level using the DsRed-EYFP-based FRET construct, making this a useful technology to monitor caspase-3 activity in living cells.     

Fluorescence depolarization dynamics of ionic strength sensors using time-resolved anisotropy

Eukaryotic cells exploit dynamic and compartmentalized ionic strength to impact a myriad of biological functions such as enzyme activities, protein-protein interactions, and catalytic functions. Herein, we investigated the fluorescence depolarization dynamics of recently developed ionic strength biosensors (mCerulean3-linker-mCitrine) in Hofmeister salt (KCl, NaCl, NaI, and Na₂SO₄) solutions. The mCerulean3-mCitrine acts as a Förster resonance energy transfer (FRET) pair, tethered together by two oppositely charged α-helices in the linker region. We developed a time-resolved fluorescence depolarization anisotropy approach for FRET analyses, in which the donor (mCerulean3) is excited by 425-nm laser pulses, followed by fluorescence depolarization analysis of the acceptor (mCitrine) in KE (lysine-glutamate), arginine-aspartate, and arginine-glutamate ionic strength sensors with variable amino acid sequences. Similar experiments were carried out on the cleaved sensors as well as an E6G2 construct, which has neutral α-helices in the linker region, as a control. Our results show distinct dynamics of the intact and cleaved sensors. Importantly, the FRET efficiency decreases and the donor-acceptor distance increases as the environmental ionic strength increases. Our chemical equilibrium analyses of the collapsed-to-stretched conformational state transition of KE reveal that the corresponding equilibrium constant and standard Gibbs free energy changes are ionic strength dependent. We also tested the existing theoretical models for FRET analyses using steady-state anisotropy, which reveal that the angle between the dipole moments of the donor and acceptor in the KE sensor are sensitive to the ionic strength. These results help establish the time-resolved depolarization dynamics of these genetically encoded donor-acceptor pairs as a quantitative means for FRET analysis, which complement traditional methods such as time-resolved fluorescence for future in vivo studies.