Self-assembled donor comprising quantum dots and fluorescent proteins for long-range fluorescence resonance energy transfer

We report on the development of a self-assembled donor for long-range fluorescence resonance energy transfer (FRET). To this end, a three-chromophore FRET (3Ch-FRET) system was constructed, which consists of a luminescent quantum dot (QD), enhanced yellow fluorescent proteins (EYFP), and Atto647-dye-modified oligonucleotides. The system was assembled by electrostatic binding of covalent EYFP-ssDNA conjugate to the QD and subsequent hybridization with complementary oligonucleotides labeled with Atto647-dye. The final conjugates comprise three different two-chromophore FRET (2Ch-FRET) subsystems, QD/EYFP, QD/Atto647, and EYFP/Atto647, respectively, which were studied in detail by steady-state and time-resolved photoluminescence measurements. The helicity of DNA allowed us to control donor/acceptor separations and thus enabled the detailed analysis of the various FRET processes. We found that the 2Ch-FRET and the 3Ch-FRET (QD/EYFP/Atto647) systems revealed FRET efficiencies and transfer rates that were affected by the availability of distinct FRET pathways. The derived energy-transfer efficiencies and F?rster radii indicated that within the 3Ch-FRET system, the 2Ch-FRET subsystem QD/EYFP showed highest FRET efficiencies ranging from 64 to 72%. Thus, it can be used as a powerful donor system that combines the intrinsic advantages of QDs (large and spectrally broad absorption cross section) and EYFP (high quantum yield) and enables long-distance FRET processes for donor-acceptor distances of up to 13 nm.     

Detection of FRET efficiency in imaging systems by photo-bleaching acceptors

Fluorescence resonance energy transfer (FRET) is widely used to obtain the distance between a donor and an acceptor in biological research. However, the detection of FRET efficiencies with fluorescence microscopy imaging systems remains a great challenge due to the difficulties of transferring gray scales of the images into fluorescence intensities, and the absence of exact quantum yields of donors and acceptors. Herein, we presented a new method to detect the FRET efficiency in imaging systems by analyzing the photo-bleaching-induced changes in fluorescent intensities of quantum dots (QDs, donors) and Cy5 dyes (acceptors). Our method is different from the previous acceptor-photo-bleaching studies in imaging systems by theoretically analyzing the bleaching process, and bringing forward a new parameter which is universal for samples of the same kind. It is convenient for calculating FRET efficiencies. There is hardly any spectral crosstalk between 605QD and Cy5, thus the FRET result is more accurate than that of many other common FRET pairs. The lengths of single-stranded and double-stranded DNA fragments in solution were determined via the analysis of FRET efficiency values. This technique provides a reliable approach to study biomacromolecules in living cells through fluorescent imaging and in situ measurements.     

Visualization of Protein‐Specific Glycosylation inside Living Cells

Protein glycosylation is a ubiquitous post‐translational modification that is involved in the regulation of many aspects of protein function. In order to uncover the biological roles of this modification, imaging the glycosylation state of specific proteins within living cells would be of fundamental importance. To date, however, this has not been achieved. Herein, we demonstrate protein‐specific detection of the glycosylation of the intracellular proteins OGT, Foxo1, p53, and Akt1 in living cells. Our generally applicable approach relies on Diels–Alder chemistry to fluorescently label intracellular carbohydrates through metabolic engineering. The target proteins are tagged with enhanced green fluorescent protein (EGFP). Förster resonance energy transfer (FRET) between the EGFP and the glycan‐anchored fluorophore is detected with high contrast even in presence of a large excess of acceptor fluorophores by fluorescence lifetime imaging microscopy (FLIM).     

Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein–protein interaction and even protein modifications in living cells. Here, we analyze the E⁰GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro . The photophysics of the two proteins is reported and shows good spectral overlap (Förster radius R ₀ = 51 Å), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration. Acceptor photobleaching (APB) and one- and two-photon fluorescence lifetime imaging microscopy (FLIM) are used to quantitatively determine FRET efficiency values. A FRET standard is introduced based on a tandem construct comprising donor and acceptor together with a 20 amino acid long cleavable peptidic linker. Reference values are obtained via enzymatic cleavage of the linker and are used as benchmarks for APB and FLIM data. E⁰GFP-mCherry shows ideal properties for FLIM detection of FRET and yields high accuracy both in vitro and in vivo . Furthermore, the recently introduced phasor approach to FLIM is shown to yield straightforward and accurate two-photon FRET efficiency data even in suboptimal experimental conditions. The consistence of these results with the reference method (both in vitro and in vivo ) reveals that this new pair can be used for very effective quantitative FRET imaging.     

Calculation of transition dipole moment in fluorescent proteins--towards efficient energy transfer

F?rster Resonance Energy Transfer (FRET) between fluorescent proteins (FPs) is widely used to construct fluorescent sensor proteins, to study intracellular protein-protein interactions and to monitor conformational changes in multidomain proteins. Although FRET depends strongly on the orientation of the transition dipole moments (TDMs) of the donor and acceptor fluorophores, this orientation dependence is currently not taken into account in FRET sensor design. Similarly, studies that use FRET to derive structural constrains typically assume a κ(2) of 2/3 or use the TDM of green fluorescent protein, as this is the only FP for which the TDM has been determined experimentally. Here we used time-dependent density functional theory (TD-DFT) methods to calculate the TDM for a comprehensive list of commonly used fluorescent proteins. The method was validated against higher levels of calculation. Validation with model compounds and the experimentally determined TDM of GFP shows that the TDM is mostly determined by the structure of the π-conjugated fluorophore and is insensitive to non-conjugated side chains or the protein surrounding. Our calculations not only provide TDM for most of the currently used FPs, but also suggest an empirical rule that can be used to obtain the TDMs for newly developed fluorescent proteins in the future.     

Are the Fluorescent Properties of the Cyan Fluorescent Protein Sensitive to Conditions of Oxidative Stress?

The modifications induced by reactive oxygen species (ROS) on fluorescent proteins (FPs) may have important implications for live cell fluorescence imaging. Using quantitative γ-radiolysis, we have studied the ROS-induced biochemical and photophysical perturbations on recombinant cyan fluorescent protein (CFP). After oxidation by the ˙OH radical, the protein displays a modified RP-HPLC elution profile, while the CFP fluorescence undergoes pronounced decreases in intensity and lifetime, without changes in its excitation and emission spectra. Meanwhile, the Förster resonant energy transfer (FRET) between the single W⁵⁷ and the chromophore remains unperturbed. These results rule out a direct oxidation of the CFP chromophore and of W⁵⁷ as well as major changes in the protein 3D structure, but show that new fluorescent forms associated to a higher level of dynamic quenching have been generated. Thus, strict in situ controls are required when CFP is to be used for FRET studies in situations of oxidative activity, or under strong illumination.     

Monitoring protein interactions in the living cell through the fluorescence decays of the cyan fluorescent protein.

Using fluorescence lifetime microspectroscopy and imaging techniques, we have studied the fluorescence of cyan fluorescent protein (CFP) transiently expressed in HEK-293 cells, in the presence or absence of its fluorescence resonance energy transfer (FRET) partner, yellow fluorescent protein (YFP). When the two proteins are attached through a 27-amino-acid linker, a 33 % average efficiency of intramolecular energy transfer is accurately determined inside the cell. Additionally, we observe a systematic quenching of the CFP fluorescence with increasing levels of protein expression. This quenching cannot be accounted for by formation of the previously described dimer of GFP-related proteins, since its magnitude is unchanged when the fluorescent proteins carry the mutation A206K shown to dissociate this dimer in vitro. Even when the intracellular protein concentration largely exceeds the in vitro dissociation constant of the dimer, self-association remains undetectable, either between free proteins or intramolecularly within the CFP-YFP construct. Instead, the detailed concentration effects are satisfactorily accounted for by a model of intermolecular, concentration-dependent energy transfer, arising from molecular proximity and crowding. In the case of CFP alone, we suggest that self-quenching could result from a pseudo-homo FRET mechanism between different, spectrally shifted emissive forms of the protein. These phenomena require careful consideration in intracellular FRET studies.     

Separating structural heterogeneities from stochastic variations in fluorescence resonance energy transfer distributions via photon distribution analysis

We establish a probability distribution analysis (PDA) method for the analysis of fluorescence resonance energy transfer (FRET) signals to determine with high precision the originating value of a shot-noise-limited signal distribution. PDA theoretical distributions are calculated explicitly including crosstalk, stochastic variations, and background and represent the minimum width that a FRET distribution must have. In this way an unambiguous distinction is made between shot-noise distributions and distributions broadened by heterogeneities. This method simultaneously and effectively extracts highly resolved information from FRET distributions. The theoretical histograms match the exact profile of histograms generated from constant transfer efficiency experimental data with a chi2 near unity. The chi2 surface suggests an ultimate level of precision with FRET of < 1% of the F?rster radius. Distributions of FRET signals in donor-acceptor-labeled DNA were unambiguously identified as being broader than shot-noise variations could explain. A model describing a Gaussian distribution of distances was tested with the PDA method and demonstrated 5 A inhomogeneities due to dye motions. The capability of this method to recover quantitative information from FRET distributions has potential applications for studying molecular conformations and dynamics. Potential sources for artifacts such as acceptor photobleaching, spectrally different observation volumes, and fluctuations of the F?rster radius are ruled out.     

Computer simulation to investigate the FRET application in DNA hybridization systems

Molecular dynamics (MD) and quantum mechanics (QM) were used to investigate fluorescence resonance energy transfer (FRET) between coumarin and ethidium in two Mergny's DNA hybridization systems. By combining the transition dipoles calculated by the quantum semi-empirical method and the conformations of the FRET probes collected by MD, FRET efficiencies were derived from the F?rster equation at five temperatures from 273 K to 313 K. The plotted efficiencies were compared with Mergny's experiments, and showed good agreement. The simulated orientation factor and isotropically averaged orientation factor were compared, and the results demonstrated that the assumption of isotropic orientations is invalid when FRET probes are close to each other. The first order kinetic assumptions were also used to calculate the transfer efficiencies, and the results show that this D-A FRET process approximates the first order kinetic reactions.     

Design strategy for a near-infrared fluorescence probe for matrix metalloproteinase utilizing highly cell permeable boron dipyrromethene

Near-infrared (NIR) fluorescence probes are especially useful for simple and noninvasive in vivo imaging inside the body because of low autofluorescence and high tissue transparency in the NIR region compared with other wavelength regions. However, existing NIR fluorescence probes for matrix metalloproteinases (MMPs), which are tumor, atherosclerosis, and inflammation markers, have various disadvantages, especially as regards sensitivity. Here, we report a novel design strategy to obtain a NIR fluorescence probe that is rapidly internalized by free diffusion and well retained intracellularly after activation by extracellular MMPs. We designed and synthesized four candidate probes, each consisting of a cell permeable or nonpermeable NIR fluorescent dye as a F?rster resonance energy transfer (FRET) donor linked to the NIR dark quencher BHQ-3 as a FRET acceptor via a MMP substrate peptide. We applied these probes for detection of the MMP activity of cultured HT-1080 cells, which express MMP2 and MT1-MMP, by fluorescence microscopy. Among them, the probe incorporating BODIPY650/665, BODIPY-MMP, clearly visualized the MMP activity as an increment of fluorescence inside the cells. We then applied this probe to a mouse xenograft tumor model prepared with HT-1080 cells. Following intratumoral injection of the probe, MMP activity could be visualized for much longer with BODIPY-MMP than with the probe containing SulfoCy5, which is cell impermeable and consequently readily washed out of the tissue. This simple design strategy should be applicable to develop a range of sensitive, rapidly responsive NIR fluorescence probes not only for MMP activity, but also for other proteases.     

Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label. R-phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.     

A FRET-Facilitated Photoswitching Using an Orange Fluorescent Protein with the Fast Photoconversion Kinetics

Fluorescent proteins photoswitchable with noncytotoxic light irradiation and spectrally distinct from multiple available photoconvertible green-to-red probes are in high demand. We have developed a monomeric fluorescent protein, called PSmOrange2, which is photoswitchable with blue light from an orange (ex./em. at 546 nm/561 nm) to a far-red (ex./em. at 619 nm/651 nm) form. Compared to another orange-to-far-red photoconvertable variant, PSmOrange2 has blue-shifted photoswitching action spectrum, 9-fold higher photoconversion contrast, and up to 10-fold faster photoswitching kinetics. This results in the 4-fold more PSmOrange2 molecules being photoconverted in mammalian cells. Compared to common orange fluorescent proteins, such as mOrange, the orange form of PSmOrange has substantially higher photostability allowing its use in multicolor imaging applications to track dynamics of multiple populations of intracellular objects. The PSmOrange2 photochemical properties allow its efficient photoswitching with common two-photon lasers and, moreover, via F?rster resonance energy transfer (FRET) from green fluorescent donors. We have termed the latter effect a FRET-facilitated photoswitching and demonstrated it using several sets of interacting proteins. The enhanced photoswitching properties of PSmOrange2 make it a superior photoconvertable protein tag for flow cytometry, conventional microscopy, and two-photon imaging of live cells.     

Biosynthesis of a fluorescent protein with extreme pseudo-Stokes shift by introducing a genetically encoded non-natural amino acid outside the fluorophore

A novel kind of fluorescent protein relying on the intramolecular interplay between two different fluorophores, one of chemical origin and one of biological origin, was developed. The fluorescent non-natural amino acid l-(7-hydroxycoumarin-4-yl)ethylglycine was site-specifically incorporated into the recombinant enhanced cyan fluorescent protein (eCFP) at a permissible surface position ～20 ? away from the protein fluorophore using amber suppression in Escherichia coli with an engineered cognate Methanococcus jannaschii tRNA synthetase. The resulting eCFP(Cou) exhibited almost quantitative intramolecular Fo?rster resonance energy transfer (FRET) between its two fluorophores, showing brilliant cyan emission at 476 nm upon excitation in the near-UV at 365 nm (a wavelength easily accessible via conventional laboratory UV sources), in contrast to its natural counterpart. Thus, this fluorescent protein with unprecedented spectroscopic properties reveals an extreme apparent Stokes shift of ～110 nm between the absorption wavelength of the coumaryl group and the emission wavelength of eCFP.     

Intramolecular energy hopping and energy trapping in polyphenylene dendrimers with multiple peryleneimide donor chromophores and a terryleneimide acceptor trap chromophore

Intramolecular F?rster-type excitation energy transfer (FRET) processes in a series of first-generation polyphenylene dendrimers substituted with spatially well-separated peryleneimide chromophores and a terryleneimide energy-trapping chromophore at the rim were investigated by steady-state and time-resolved fluorescence spectroscopy. Energy-hopping processes among the peryleneimide chromophores are revealed by anisotropy decay times of 50--80 ps consistent with a FRET rate constant of k(hopp) = 4.6 ns(-1). If a terryleneimide chromophore is present at the rim of the dendrimer together with three peryleneimide chromophores, more than 95% of the energy harvested by the peryleneimide chromophores is transferred and trapped in the terryleneimide. The two decay times (tau(1) = 52 ps and tau(2) = 175 ps) found for the peryleneimide emission band are recovered as rise times at the terryleneimide emission band proving that the energy trapping of peryleneimide excitation energy by the terryleneimide acceptor occurs via two different, efficient pathways. Molecular- modeling-based structures tentatively indicate that the rotation of the terryleneimide acceptor group can lead to a much smaller distance to a single donor chromophore, which could explain the occurrence of two energy-trapping rate constants. All energy-transfer processes are quantitatively describable with F?rster energy transfer theory, and the influence of the dipole orientation factor in the F?rster equation is discussed.     

Phosphorescence Energy Transfer: Ambient Afterglow Fluorescence from Water‐Processable and Purely Organic Dyes via Delayed Sensitization

Ambient afterglow luminescence from metal‐free organic chromophores would provide a promising alternative to the well‐explored inorganic phosphors. However, the realization of air‐stable and solution‐processable organic afterglow systems with long‐lived triplet or singlet states remains a formidable challenge. In the present study, a delayed sensitization of the singlet state of organic dyes via phosphorescence energy transfer from organic phosphors is proposed as an alternative strategy to realize “afterglow fluorescence”. This concept is demonstrated with a long‐lived phosphor as the energy donor and commercially available fluorescent dyes as the energy acceptor. Triplet‐to‐singlet Förster‐resonance energy‐transfer (TS‐FRET) between donor and acceptor chromophores, which are co‐organized in an amorphous polymer matrix, results in tuneable yellow and red afterglow from the fluorescent acceptors. Moreover, these afterglow fluorescent hybrids are highly solution‐processable and show excellent air‐stability with good quantum yields.     

Fluorescence resonance energy transfer between quantum dot donors and dye-labeled protein acceptors

We used luminescent CdSe-ZnS core-shell quantum dots (QDs) as energy donors in fluorescent resonance energy transfer (FRET) assays. Engineered maltose binding protein (MBP) appended with an oligohistidine tail and labeled with an acceptor dye (Cy3) was immobilized on the nanocrystals via a noncovalent self-assembly scheme. This configuration allowed accurate control of the donor-acceptor separation distance to a range smaller than 100 A and provided a good model system to explore FRET phenomena in QD-protein-dye conjugates. This QD-MBP conjugate presents two advantages: (1) it permits one to tune the degree of spectral overlap between donor and acceptor and (2) provides a unique configuration where a single donor can interact with several acceptors simultaneously. The FRET signal was measured for these complexes as a function of both degree of spectral overlap and fraction of dye-labeled proteins in the QD conjugate. Data showed that substantial acceptor signals were measured upon conjugate formation, indicating efficient nonradiative exciton transfer between QD donors and dye-labeled protein acceptors. FRET efficiency can be controlled either by tuning the QD photoemission or by adjusting the number of dye-labeled proteins immobilized on the QD center. Results showed a clear dependence of the efficiency on the spectral overlap between the QD donor and dye acceptor. Apparent donor-acceptor distances were determined from efficiency measurements and corresponding F?rster distances, and these results agreed with QD bioconjugate dimensions extracted from structural data and core size variations among QD populations.     

Fluorescence measurements of duplex DNA oligomers under conditions conducive for forming M-DNA (a metal-DNA complex)

M-DNA (a metal complex of DNA with millimolar concentrations of Zn2+, Co2+, or Ni2+ and basic pH) has been proposed to undergo electron transfer over long distances along the helix and has generated interest as a potential building block for nanoelectronics. We show that DNA aggregates form under solvent conditions favorable for M-DNA (millimolar zinc and pH = 8.6) by fluorescence correlation spectroscopy. We have performed steady-state F?rster resonance energy transfer (FRET) experiments with DNA oligomers conjugated with 6-carboxyfluorescein and tetramethylrhodamine to the opposite ends of double-stranded DNA (dsDNA) molecules. Enhanced acceptor emission is observed for distances larger than expected for identical DNA molecules with no zinc. To avoid intermolecular FRET, the fluorescently labeled dsDNA is diluted with a 100-fold excess of unlabeled dsDNA. The intramolecular FRET efficiency increases 25-fold for a 30-mer doubly labeled duplex DNA molecule upon addition of millimolar concentrations of zinc ions. Without zinc, this oligomer has less than 1% FRET efficiency. This dramatic increase in the FRET efficiency points to either significant changes in the F?rster radius or fraying of the ends of the DNA helices. The latter hypothesis is supported by our experiments with a 9-mer that show dissociation of the duplex by zinc ions.     

A 10-A spectroscopic ruler applied to short polyprolines

Fluorescence resonance energy transfer (FRET) from the amino acid tryptophan (Trp) as donor and a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as acceptor in peptides of the general structure Trp-(Pro)n-Dbo-NH2 (n = 1-6) was investigated by steady-state and time-resolved fluorescence, CD, and NMR spectroscopy as well as by molecular dynamics (MD) simulations (GROMOS96 force field). The Trp/Dbo FRET pair is characterized by a very short F?rster radius (R0 ca. 9 A), which allowed distance determinations in such short peptides. Water and propylene glycol were investigated as solvents. The peptides were designed to show an early nucleation of the poly(Pro)II (PPII) secondary helix structure for n > or = 2, which was confirmed by their CD spectra. The shortest peptide (n = 1) adopts preferentially the trans conformation about the Trp-Pro bond, as confirmed by NMR spectra. The FRET efficiencies ranged 2-72% and were found to depend sensitively on the peptide length, i.e., the number of intervening proline residues. The analysis of the FRET data at different levels of theory (assuming either a fixed distance or distance distributions according to a wormlike chain or Gaussian model) afforded donor-acceptor distances between ca. 8 A (n = 1) and ca. 16 A (n = 6) in water, which were found to be similar or slightly higher in propylene glycol. The distances afforded by the Trp/Dbo FRET pair were found to be reasonable in comparison to literature data, expectations from the PPII helix structure, and the results from MD simulations. The persistence lengths for the longer peptides were found to lie at 30-70 A in water and 220 +/- 40 A in propylene glycol, suggesting a more rigid PPII helical structure in propylene glycol. A detailed comparison with literature data on FRET in polyprolines demonstrates that the donor-acceptor distances extracted by FRET are correlated with the F?rster radii of the employed FRET pairs. This demonstrates the limitations of using FRET as a spectroscopic ruler for short polyprolines, which is presumably due to the breakdown of the point dipole approximation in F?rster theory, when the size of the chromophores becomes comparable or larger than the distances under investigation.     

Homogeneous time resolved fluorescence resonance energy transfer using rare earth cryptates as a tool for probing molecular interactions in biology

A homogeneous assay technology using time resolved fluorescence and fluorescence resonance energy transfer is described. A new class of fluorescent complexes, the cryptates, have been used as fluorescent donor with cross-linked allophycocyanin as acceptor. This new donor/acceptor shows an exceptionally high F?rster distance R0 of 9 nm. This allows to build up a set of strategies to probe the interactions of biomolecules in biology, particularly for high throughput screening applications. In this article, we describe the basics of the technology and review applications developed for studying different key molecular interactions involved in cellular processes.