Förster Resonance Energy Transfer Method Research Articles

Structured illumination-based super-resolution live-cell quantitative FRET imaging

Förster resonance energy transfer (FRET) microscopy provides unique insight into the functionality of biological systems via imaging the spatiotemporal interactions and functional state of proteins. Distinguishing FRET signals from sub-diffraction regions requires super-resolution (SR) FRET imaging, yet is challenging to achieve from living cells. Here, we present an SR FRET method named SIM-FRET that combines SR structured illumination microscopy (SIM) imaging and acceptor sensitized emission FRET imaging for live-cell quantitative SR FRET imaging. Leveraging the robust co-localization prior of donor and accepter during FRET, we devised a mask filtering approach to mitigate the impact of SIM reconstruction artifacts on quantitative FRET analysis. Compared to wide-field FRET imaging, SIM-FRET provides nearly twofold spatial resolution enhancement of FRET imaging at sub-second timescales and maintains the advantages of quantitative FRET analysis in vivo. We validate the resolution enhancement and quantitative analysis fidelity of SIM-FRET signals in both simulated FRET models and live-cell FRET-standard construct samples. Our method reveals the intricate structure of FRET signals, which are commonly distorted in conventional wide-field FRET imaging.

Detecting RNA–Protein Interactions With EGFP‐Cy3 FRET by Acceptor Photobleaching

Förster Resonance Energy Transfer (FRET) is a great tool for cell biologists to investigate molecular interactions in live specimens. FRET is a distance-dependent phenomenon which can detect molecular interactions at distances between 1-10 nm. Several FRET approaches are reported in the literature for live and fixed cells to study protein-protein interactions; this protocol provides details of acceptor photobleaching as a FRET method to study RNA-Protein interactions. Cy3-labeled RNA foci (FRET acceptors) are photobleached at the intra-cellular site of interest (the nuclei) and the intensity of the EGFP-tagged proteins (FRET donors) at that same site are measured pre- and post- photobleaching. In principle, FRET is detected if the intensity of EGFP increases after photobleaching of Cy3. This protocol describes necessary steps and appropriate controls to conduct FRET measurements by the acceptor photobleaching method. Successful applications of this protocol will provide data to support the conclusion that EGFP-labeled proteins directly interact with Cy3-labeled RNA at the site of photobleaching. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: FRET in fixed cells Alternate Protocol: FRET in live cells.

Diffusion-Enhanced Förster Resonance Energy Transfer in Flexible Peptides: From the Haas-Steinberg Partial Differential Equation to a Closed Analytical Expression.

In the huge field of polymer structure and dynamics, including intrinsically disordered peptides, protein folding, and enzyme activity, many questions remain that cannot be answered by methodology based on artificial intelligence, X-ray, or NMR spectroscopy but maybe by fluorescence spectroscopy. The theory of Förster resonance energy transfer (FRET) describes how an optically excited fluorophore transfers its excitation energy through space to an acceptor moiety-with a rate that depends on the distance between donor and acceptor. When the donor and acceptor moiety are conjugated to different sites of a flexible peptide chain or any other linear polymer, the pair could in principle report on chain structure and dynamics, on the site-to-site distance distribution, and on the diffusion coefficient of mutual site-to-site motion of the peptide chain. However, the dependence of FRET on distance distribution and diffusion is not defined by a closed analytical expression but by a partial differential equation (PDE), by the Haas-Steinberg equation (HSE), which can only be solved by time-consuming numerical methods. As a second complication, time-resolved FRET measurements have thus far been deemed necessary. As a third complication, the evaluation requires a computationally demanding but indispensable global analysis of an extended experimental data set. These requirements have made the method accessible to only a few experts. Here, we show how the Haas-Steinberg equation leads to a closed analytical expression (CAE), the Haas-Steinberg-Jacob equation (HSJE), which relates a diffusion-diagnosing parameter, the effective donor-acceptor distance, to the augmented diffusion coefficient, J, composed of the diffusion coefficient, D, and the photophysical parameters that characterize the used FRET method. The effective donor-acceptor distance is easily retrieved either through time-resolved or steady-state fluorescence measurements. Any global fit can now be performed in seconds and minimizes the sum-of-square difference between the experimental values of the effective distance and the values obtained from the HSJE. In summary, the HSJE can give a decisive advantage in applying the speed and sensitivity of FRET spectroscopy to standing questions of polymer structure and dynamics.

Analysis insights for three FRET pairs of chemically unlinked two-molecule FRET cytometry.

Förster resonance energy transfer (FRET) is the direct energy exchange between two-component fluorescent molecules. FRET methods utilize chemically linked molecules or unlinked fluorescent molecules such as fluoresscent protein-protein interactions. FRET is therefore a powerful indicator of molecular proximity, but standardized determination of FRET efficiency is challenged when investigating natural (chemically unlinked) interactions. In this paper, we have examined the interactions of tumor necrosis factor receptor-1 (TNFR1) molecules expressed as recombinant C-terminal fusion proteins of cyan, yellow, or red fluorescent protein (-CFP, -YFP, or -RFP) to evaluate two-molecule chemically unlinked FRET by flow cytometry. We demonstrate three independent FRET pairs of TNFR1 CFP→YFP (FRET-1), YFP→RFP (FRET-2) and CFP→RFP (FRET-3), by comparing TNFR1+TNFR1 with non-interacting TNFR1+CD27 proteins, on both LSR-II and Fortessa X-20 cytometers. We describe genuine FRET activities reflecting TNFR1 homotypic interactions. The FRET events can be visualized during sample acquisition via the use of "spiked" FRET donor cells, together with TNFR1+TNFR1 co-transfected cells, as FRET channel mean fluorescence intensity (MFI) overlays. FRET events can also be indicated by comparing concatenated files of cells expressing either FRET positive events (TNFR1+TNFR1) or FRET negative events (TNFR1+CD27) to generate single-cell scatter plots showing loss of FRET donor brightness. Robust determination of FRET efficiency is then confirmed at the single-cell level by applying matrix calculations based on the measurements of FRET, using donor, acceptor, and FRET fluorescent intensities (I), detector channel emission coefficient (S), fluorescent protein extinction coefficients (ε) and the α factor. In this TNFR1-based system the mean CFP→YFP FRET-1 efficiency is 0.43 (LSR-II) and 0.41 (Fortessa X-20), the mean YFP→RFP FRET-2 efficiency is 0.30 (LSR-II) and 0.29 (Fortessa X-20), and the mean CFP→RFP FRET-3 efficiency is 0.56 (LSR-II) and 0.54 (Fortessa X-20). This study also embraces multi-dimensional clustering using t-SNE, Fit-SNE, UMAP, Tri-Map and PaCMAP to further demonstrate FRET. These approaches establish a robust system for standardized detection of chemically unlinked TNFR1 homotypic interactions with three individual FRET pairs.

J‐Aggregate‐Based FRET Monitoring of Drug Release from Polymer Nanoparticles with High Drug Loading

AbstractUnderstanding drug‐release kinetics is critical for the development of drug‐loaded nanoparticles. We developed a J‐aggregate‐based Förster‐resonance energy‐transfer (FRET) method to investigate the release of novel high‐drug‐loading (50 wt %) nanoparticles in comparison with low‐drug‐loading (0.5 wt %) nanoparticles. Single‐dye‐loaded nanoparticles form J‐aggregates because of the high dye‐loading (50 wt %), resulting in a large red‐shift (≈110 nm) in the fluorescence spectrum. Dual‐dye‐loaded nanoparticles with high dye‐loading using FRET pairs exhibited not only FRET but also a J‐aggregate red‐shift (116 nm). Using this J‐aggregate‐based FRET method, dye‐core–polymer‐shell nanoparticles showed two release processes intracellularly: the dissolution of the dye aggregates into dye molecules and the release of the dye molecules from the polymer shell. Also, the high‐dye‐loading nanoparticles (50 wt %) exhibited a slow release kinetics in serum and relatively quick release in cells, demonstrating their great potential in drug delivery.

Photoswitching FRET Studies of Doxorubicin-Chromatin Interactions

The molecular basis for the action of anti-cancer drugs is of interest for further drug development as well as for better understanding of adverse side effects. Doxorubicin is a DNA intercalating anti-cancer drug thought to work by inhibition of topoisomerase II leading to double-strand breaks and subsequent cancer cell death. It is also proposed to display a novel mechanism of action which results in eviction of some core histones from chromatin and leads to deregulation of the cancer cell transcriptome. Uncommon amongst anti-cancer drugs, many anthracyclines such as doxorubicin have absorption spectra in the visible range which allows Förster Resonance Energy Transfer (FRET) studies of their interactions with chromatin. Since Fluorescence Lifetime Imaging Microscopy (FLIM) FRET studies do not require an acceptor to be fluorescent to measure energy transfer, previous works concentrated on doxorubicin-chromatin interactions within intact tumors by imaging the fluorescence lifetimes of EGFP labeled histone H1 or H2B. Our previously developed photoswitching FRET (psFRET) method mimics the advantage of FLIM by also not requiring a fluorescent acceptor. This property allows us to monitor energy transfer between doxorubicin and the core histone proteins tagged with the photoswitchable fluorescent protein, Dronpa. By tagging the N or C termini of the core histones, the Dronpa protein can be placed at multiple different positions on the periphery of a nucleosome and thus allow a survey of FRET efficiencies from numerous points. Here, we present psFRET studies in our efforts to better define binding sites for doxorubicin as well as its potential for histone eviction from chromatin.

FRET in a Polymeric Nanocarrier: IR-780 and IR-780-PDMS.

We introduce a method to monitor the integrity of micellar nanocarriers using a novel fluorescent dye, IR-780-PDMS and Förster resonance energy transfer (FRET) as a readout. In addition, these dye-loaded nanocarriers can be used as a phototoxic agent in vitro. Mainly, a nanocarrier was designed, based on a previously described amphiphilic ABA-copolymer (Pip-PMOXA-PDMS-PMOXA-Pip) scaffold that incorporates the fluorescent FRET dye partners IR-780-PDMS (donor) and IR-780 (acceptor). The confirmation of FRET (that only occurs when donor and acceptor are in the required close proximity of less than ∼10 nm) in the nanocarriers is used to prove that the acceptor dye (IR-780) is still contained in its hydrophobic core. We measured such FRET signals of the nanocarriers also upon cellular uptake into HeLa cells using fluorescence-lifetime imaging microscopy (FLIM). Confocal laser scanning microscopy after incubation with nanocarriers demonstrated the intracellular uptake of the particles and their localization in an intracellular granular pattern. To demonstrate the intactness of the nanocarriers by detection of FRET we measured the fluorescence lifetime (FLIM) of the donor dye. FLIM showed that both types of lifetimes, that of the quenched donor, and that of the unquenched donor were present, in a granular pattern and homogeneously in the cytosol, respectively, indicating the presence of intracellular intact and disintegrated micellar nanocarriers. These data show that the herewith-described FRET method allows monitoring the intactness of nanocarriers while en route to the target, and also that the cargo is delivered and released within a potential target cell. In addition, near-infrared (NIR) irradiation of IR-780-loaded micellar nanocarriers leads to photocytotoxicity, which we demonstrated in in vitro experiments. Our findings open potential avenues in photodynamic therapy (PDT) of cancer.

FRET at the Single Molecule Level using Molecular Brightness and Fluorescence Correlation Spectroscopy

Förster resonance energy transfer (FRET) is a noninvasive, quantitative method to study intermolecular interactions and conformational changes. The energy transfer efficiency of FRET pairs are routinely quantified using steady-state fluorescence spectroscopy or time-resolved fluorescence for which the main challenge of these approaches is the averaging over a larger ensemble of molecules. Here, we present a new approach for determining the energy transfer efficiency of hetero-FRET probes at the single-molecule level, using time-resolved fluorescence correlation spectroscopy (FCS) and molecular brightness (i.e., the number of fluorescence photons per molecule emitted while diffusing in the open observation volume). In this approach, the molecular brightness of the donor, in the presence and absence of the acceptor, is related to the energy transfer efficiency. We used the engineered hetero-FRET probe, RD, which consists of a mCerulean-linker-mCitrine construct, as the model system. Laser intensity-dependent FCS experiments were carried out on both the intact and cleaved RD indicating an energy transfer efficiency of (50 ± 10)% for RD in buffer, corresponding to a distance of 7 ± 2 nm between the donor and acceptor. These results demonstrate that our FCS approach is applicable to hetero-FRET constructs in well-controlled environments. Further it should be useful in living cells because it has the added advantage of requiring low expression levels of the FRET sensor, in contrast to more conventional FRET methods.

Fluorescence lifetime based distance measurement illustrates conformation changes of PYL10-CL2 upon ABA binding in solution state

Förster resonance energy transfer (FRET) is a widely used distance measurement method to illustrate protein conformational dynamics. The FRET method relies on the distance between donor and acceptor, as well as the labelling efficiency, the size and the properties of the fluorophores. Here, we labelled a pair of small fluorophores and calculated the energy transferred efficiency through fluorescence lifetime analysis, which can provide more reliable distance measurement than intensity attenuation. The donor fluorophore, 7-hydroxycoumarin-4-yl-ethylglycine (HC), was genetically incorporated into specific sites of PYL10, obtaining complete labelling efficiency. The acceptor fluorophore, Alexa488, was labelled through the disulfide bond, whose labelling efficiency was estimated through both absorption peaks and lifetime populations. Fluorescence lifetime and anisotropy analysis showed ABA-induced local conformation changes and dynamics of several HC incorporation sites of PYL10. The lifetime-based FRET distance measurement illustrated the conformation changes of PYL10 with or without ABA application, which is consistent with the previously reported crystal structures.

Homotransfer of FRET Reporters for Live Cell Imaging.

Förster resonance energy transfer (FRET) between fluorophores of the same species was recognized in the early to mid-1900s, well before modern heterotransfer applications. Recently, homotransfer FRET principles have re-emerged in biosensors that incorporate genetically encoded fluorescent proteins. Homotransfer offers distinct advantages over the standard heterotransfer FRET method, some of which are related to the use of fluorescence polarization microscopy to quantify FRET between two fluorophores of identical color. These include enhanced signal-to-noise, greater compatibility with other optical sensors and modulators, and new design strategies based upon the clustering or dimerization of singly-labeled sensors. Here, we discuss the theoretical basis for measuring homotransfer using polarization microscopy, procedures for data collection and processing, and we review the existing genetically-encoded homotransfer biosensors.

Two Orders of Magnitude Variation of Diffusion-Enhanced Förster Resonance Energy Transfer in Polypeptide Chains.

A flexible peptide chain displays structural and dynamic properties that correspond to its folding and biological activity. These properties are mirrored in intrachain site-to-site distances and diffusion coefficients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of Förster resonance energy transfer (FRET) between two sites labeled with a FRET donor and acceptor. The relatively large Förster radii of traditional FRET methods (R0 > 20 Å) lead to a fairly low contribution of diffusion. We introduced short-distance FRET (sdFRET) where Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, acts as acceptor paired with donors, such as naphtylalanine (NAla), tryptophan, 5-l-fluorotryptophan, or tyrosine. The Förster radii are always close to 10 Å, which makes sdFRET highly sensitive to diffusional motion. We recently found indications that the FRET enhancement caused by diffusion depends symmetrically on the product of the radiative fluorescence lifetime of the donor and the diffusion coefficient. In this study, we varied this product by two orders of magnitude, using both donors of different lifetime, NAla and FTrp, as well as a varying viscogen concentration, to corroborate this statement. We demonstrate the consequences of this relationship in evaluating the impact of viscogenic coadditives on peptide dimensions.

Conformational Changes Spanning Angstroms to Nanometers via a Combined Protein-Induced Fluorescence Enhancement-Förster Resonance Energy Transfer Method.

Förster resonance energy transfer (FRET)-based single-molecule techniques have revolutionized our understanding of conformational dynamics in biomolecular systems. Recently, a new single-molecule technique based on protein-induced fluorescence enhancement (PIFE) has aided studies in which minimal (<3 nm) displacements occur. Concerns have been raised regarding whether donor fluorophore intensity (and correspondingly fluorescence quantum yield Φf) fluctuations, intrinsic to PIFE methods, may adversely affect FRET studies when retrieving the donor-acceptor dye distance. Here, we initially show through revisions of Förster's original equation that distances may be calculated in FRET experiments regardless of protein-induced intensity (and Φf) fluctuations occurring in the donor fluorophore. We additionally demonstrate by an analysis of the recorded emission intensity and competing decay pathways that PIFE and FRET methods may be conveniently combined, providing parallel complementary information in a single experiment. Single-molecule studies conducted with Cy3- and ATTO647N-labeled RNA structures and the HCV-NS5B polymerase protein undergoing binding dynamics along the RNA backbone provide a case study to validate the results. The analysis behind the proposed method enables for PIFE and FRET changes to be disentangled when both FRET and PIFE fluctuate over time following protein arrival and, for example, sliding. A new method, intensity-FRET, is thus proposed to monitor conformational changes spanning from angstroms to nanometers.

Accurate Determination of the RNA Three-Way Junctions Via Single-Molecule High-Precision Fret Measurements

In nature RNA helices are often linked via various junctions and bulges that determine overall 3D structure which are used as building blocks and functional components in nanotechnology applications. Hence visualization of RNA conformations is crucial for understanding their biological functions. Förster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling can be used to determine the structure of these junctions. Multi-parameter fluorescence detection (MFD) of single molecules and ensemble Time-Correlated Single Photon Counting (eTCSPC) measurements were applied to perform FRET study on systematic series of 6 RNA three- way-junctions (3WJs) derived from the hairpin ribozyme. Bulge and sequence variations were considered as dominant factors influencing junction, two of which have bulge (two and five unpaired nucleotides) in the junction region. We have generated a database of overall 252 different RNA 3WJs labeled with Alexa488 and Cy5 fluorescent dyes. The analysis toolkit included probability distribution analysis (PDA) for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation. We detected only one predominant conformer for RNAs 3WJ. Furthermore we report that bulges in the junction determine orientation and rotation of helices, inducing coaxial stacking. We provide a geometrical description of the junction geometry and our results show that small changes in the sequence make dramatic changes in RNA 3WJ tertiary structures which are expected to have significant impact on the functionality. As the accuracy of FRET restrained modeling depends on the correct description of the dye behavior, we characterized the position dependent fluorescent properties of the coupled dyes and statistically analyzed the systematic deviations of the calculated FRET distances from the model generated distances. This allowed us to identify the problematic labeling positions. Thus we could improve the accuracy of the FRET method by careful selection of labeling sites.

FRET Imaging in Three-dimensional Hydrogels.

Imaging of Förster resonance energy transfer (FRET) is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

FRET Imaging in Three-dimensional Hydrogels

Imaging of Förster resonance energy transfer (FRET) is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

Affinity assisted selection of antibodies for Point of Care TSH immunoassay with limited wash

BackgroundMolecular binding characteristics of several thyroid stimulating hormone (TSH) antibodies were determined for the TSH antigen, along with its closely related endogenous interfering hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG). MethodsThis data was compared to the same antibodies used in the low wash sandwich ELISA immunoassay system, the Point of Care i-STAT® immunoassay. From this information we developed binding criteria useful in the low wash i-STAT® immunoassay to permit good signal generation from TSH and low cross-reactivity from its interfering hormones. For the TSH Assay we have developed characteristics that enable antibody selection in the i-STAT® immunoassay cartridge. Our antibody screening approach used a dot blot approach as a first screen to select for the most useful antibodies. We then compared a FRET (Förster Resonance Energy Transfer) and electrochemical cartridge approach to determine the appropriate antibody combinations. ResultsBoth methods generated similar data, but the FRET method was not capable of differentiating the antibody with the best characteristics as a capture antibody or a detection conjugate in a sandwich ELISA assay. Finally, we performed binding characterizations of the antibodies using each of the above mentioned glycoproteins. ConclusionsWe found that we need sub-picomolar detection of TSH, and at least 100 fold or higher values for the cross-reacting species.

Maximizing the quantitative accuracy and reproducibility of Förster resonance energy transfer measurement for screening by high throughput widefield microscopy

Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) provides insights into the proximities and orientations of FPs as surrogates of the biochemical interactions and structures of the factors to which the FPs are genetically fused. As powerful as FRET methods are, technical issues have impeded their broad adoption in the biologic sciences. One hurdle to accurate and reproducible FRET microscopy measurement stems from variable fluorescence backgrounds both within a field and between different fields. Those variations introduce errors into the precise quantification of fluorescence levels on which the quantitative accuracy of FRET measurement is highly dependent. This measurement error is particularly problematic for screening campaigns since minimal well-to-well variation is necessary to faithfully identify wells with altered values. High content screening depends also upon maximizing the numbers of cells imaged, which is best achieved by low magnification high throughput microscopy. But, low magnification introduces flat-field correction issues that degrade the accuracy of background correction to cause poor reproducibility in FRET measurement. For live cell imaging, fluorescence of cell culture media in the fluorescence collection channels for the FPs commonly used for FRET analysis is a high source of background error. These signal-to-noise problems are compounded by the desire to express proteins at biologically meaningful levels that may only be marginally above the strong fluorescence background. Here, techniques are presented that correct for background fluctuations. Accurate calculation of FRET is realized even from images in which a non-flat background is 10-fold higher than the signal.

Analysis of steady-state Förster resonance energy transfer data by avoiding pitfalls: Interaction of JAK2 tyrosine kinase with N-methylanthraniloyl nucleotides

Förster resonance energy transfer (FRET) between the fluorescent ATP analogue 2′/3′-(N-methyl-anthraniloyl)-adenosine-5′-triphosphate (MANT–ATP) and enzymes is widely used to determine affinities for ATP–protein binding. However, in analysis of FRET fluorescence data, several important parameters are often ignored, resulting in poor accuracy of the calculated dissociation constant (Kd). In this study, we systematically analyze factors that interfere with Kd determination and describe methods for correction of primary and secondary inner filter effects that extend the use of the FRET method to higher MANT nucleotide concentrations. The interactions of the fluorescent nucleotide analogues MANT–ATP, MANT–ADP [2′/3′-O-(N-methylanthraniloyl) adenosine diphosphate], and MANT–AMP [2′/3′-O-(N-methylanthraniloyl) adenosine monophosphate] with the JAK2 tyrosine kinase domain are characterized. Taking all interfering factors into consideration, we found that JAK2 binds MANT–ATP tightly with a Kd of 15 to 25nM and excluded the presence of a second binding site. The affinity for MANT–ADP is also tight with a Kd of 50 to 80nM, whereas MANT–AMP does not bind. Titrations of JAK2 JH1 with nonhydrolyzable ATP analogue MANT–ATP-γ-S [2′/3′-O-(N-methylanthraniloyl) adenosine-5′-(thio)- triphosphate] yielded a Kd of 30 to 50nM. The methods demonstrated here are applicable to other enzyme–fluorophore combinations and are expected to help improve the analysis of steady-state FRET data in MANT nucleotide binding studies and to obtain more accurate results for the affinities of nucleotide binding proteins.

Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells.

The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs.

Quantitative analysis of FRET assay in biology—new developments in protein interaction affinity and protease kinetics determinations in the SUMOylation cascade

Förster resonance energy transfer (FRET) techniques have been widely used in biological studies in vitro and in vivo and are powerful tools for elucidating protein interactions in many regulatory cascades. FRET occurs between oscillating dipoles of two fluorophores with overlapping emission and excitation wavelengths and is dependent on the spectroscopic and geometric properties of the donor-acceptor pair. Various efforts have been made to develop quantitative FRET methods to accurately determine the interaction affinity and kinetics parameters. SUMOylation is an important post-translational protein modification with key roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENP) act as endopeptidases to process the pre-SUMO or an isopeptidase to deconjugate SUMO from its substrate. Here we also summarize recent developments of theoretical and experimental procedures for determining the protein interaction dissociation constant, K d, and protease kinetics parameters, k cat and K m, in the SUMOylation pathway. The general principles of these quantitative FRET-based measurements can be applied to other protein interactions and proteases.