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1.  Classic Maximum Entropy Recovery of the Average Joint Distribution of Apparent FRET Efficiency and Fluorescence Photons for Single-molecule Burst Measurements 
The Journal of Physical Chemistry. B  2012;116(13):4006-4015.
We describe a method for analysis of single-molecule Förster resonance energy transfer (FRET) burst measurements using classic maximum entropy. Classic maximum entropy determines the Bayesian inference for the joint probability describing the total fluorescence photons and the apparent FRET efficiency. The method was tested with simulated data and then with DNA labeled with fluorescent dyes. The most probable joint distribution can be marginalized to obtain both the overall distribution of fluorescence photons and the apparent FRET efficiency distribution. This method proves to be ideal for determining the distance distribution of FRET-labeled biomolecules, and it successfully predicts the shape of the recovered distributions.
PMCID: PMC3320690  PMID: 22338694
Single-molecule FRET; Bayesian analysis; photon distribution analysis; maximum entropy
2.  Graphical models for inferring single molecule dynamics 
BMC Bioinformatics  2010;11(Suppl 8):S2.
The recent explosion of experimental techniques in single molecule biophysics has generated a variety of novel time series data requiring equally novel computational tools for analysis and inference. This article describes in general terms how graphical modeling may be used to learn from biophysical time series data using the variational Bayesian expectation maximization algorithm (VBEM). The discussion is illustrated by the example of single-molecule fluorescence resonance energy transfer (smFRET) versus time data, where the smFRET time series is modeled as a hidden Markov model (HMM) with Gaussian observables. A detailed description of smFRET is provided as well.
The VBEM algorithm returns the model’s evidence and an approximating posterior parameter distribution given the data. The former provides a metric for model selection via maximum evidence (ME), and the latter a description of the model’s parameters learned from the data. ME/VBEM provide several advantages over the more commonly used approach of maximum likelihood (ML) optimized by the expectation maximization (EM) algorithm, the most important being a natural form of model selection and a well-posed (non-divergent) optimization problem.
The results demonstrate the utility of graphical modeling for inference of dynamic processes in single molecule biophysics.
PMCID: PMC2966289  PMID: 21034427
3.  Visualization of Protein Interactions in Living Cells 
Self Nonself  2011;2(2):98-107.
Ligand binding to cell membrane receptors sets off a series of protein interactions that convey the nuances of ligand identity to the cell interior. The information may be encoded in conformational changes, the interaction kinetics and, in the case of multichain immunoreceptors, by chain rearrangements. The signals may be modulated by dynamic compartmentalization of the cell membrane, cellular architecture, motility, and activation—all of which are difficult to reconstitute for studies of receptor signaling in vitro. In this paper, we will discuss how protein interactions in general and receptor signaling in particular can be studied in living cells by different fluorescence imaging techniques. Particularly versatile are methods that exploit Förster resonance energy transfer (FRET), which is exquisitely sensitive to the nanometer-range proximity and orientation between fluorophores. Fluorescence correlation microscopy (FCM) can provide complementary information about the stoichiometry and diffusion kinetics of large complexes, while bimolecular fluorescence complementation (BiFC) and other complementation techniques can capture transient interactions. A continuing challenge is extracting from the imaging data the quantitative information that is necessary to verify different models of signal transduction.
PMCID: PMC3268995  PMID: 22299061
4.  Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets 
PLoS ONE  2013;8(8):e70687.
Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.
PMCID: PMC3734241  PMID: 23940626
5.  Internal Calibration Förster Resonance Energy Transfer Assay: A Real-Time Approach for Determining Protease Kinetics 
Sensors (Basel, Switzerland)  2013;13(4):4553-4570.
Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research. This powerful tool can elucidate protein interactions in either a dynamic or steady state. We recently developed a series of FRET-based technologies to determine protein interaction dissociation constant and for use in high-throughput screening assays of SUMOylation. SUMO (small ubiquitin-like modifier) is conjugated to substrates through an enzymatic cascade. This important posttranslational protein modification is critical for multiple biological processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate SUMO from its substrate. Here, we describe a novel quantitative FRET-based protease assay for determining the kinetics of SENP1. Our strategy is based on the quantitative analysis and differentiation of fluorescent emission signals at the FRET acceptor emission wavelengths. Those fluorescent emission signals consist of three components: the FRET signal and the fluorescent emissions of donor (CyPet) and acceptor (YPet). Unlike our previous method in which donor and acceptor direct emissions were excluded by standard curves, the three fluorescent emissions were determined quantitatively during the SENP digestion process from onesample. New mathematical algorithms were developed to determine digested substrate concentrations directly from the FRET signal and donor/acceptor direct emissions. The kinetic parameters, kcat, KM, and catalytic efficiency (kcat/KM) of SENP1 catalytic domain for pre-SUMO1/2/3 were derived. Importantly, the general principles of this new quantitative methodology of FRET-based protease kinetic determinations can be applied to other proteases in a robust and systems biology approach.
PMCID: PMC3673099  PMID: 23567524
quantitative FRET analysis; internal calibration; one-sample assay; protease kinetics; SENP
6.  Development of FRET Assay into Quantitative and High-throughput Screening Technology Platforms for Protein–Protein Interactions 
Annals of Biomedical Engineering  2010;39(4):1224-1234.
Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a very powerful tool in elucidating protein interactions in many cellular processes. Ubiquitination and SUMOylation are multi-step cascade reactions, involving multiple enzymes and protein–protein interactions. Here we report the development of dissociation constant (Kd) determination for protein–protein interaction and cell-based high-throughput screening (HTS) assay in SUMOylation cascade using FRET technology. These developments are based on steady state and high efficiency of fluorescent energy transfer between CyPet and YPet fused with SUMO1 and Ubc9, respectively. The developments in theoretical and experimental procedures for protein interaction Kd determination and cell-based HTS provide novel tools in affinity measurement and protein interaction inhibitor screening. The Kd determined by FRET between SUMO1 and Ubc9 is compatible with those determined with other traditional approaches, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). The FRET-based HTS is pioneer in cell-based HTS. Both Kd determination and cell-based HTS, carried out in 384-well plate format, provide powerful tools for large-scale and high-throughput applications.
PMCID: PMC3069323  PMID: 21174150
SUMOylation; Förster resonance energy transfer; Kd affinity determination; High-throughput screening
7.  Fluorescence Polarization and Fluctuation Analysis Monitors Subunit Proximity, Stoichiometry, and Protein Complex Hydrodynamics 
PLoS ONE  2012;7(5):e38209.
Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1–10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9±1.2 subunit, but values ranged from 10–14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2±1.3 subunits in the holoenzyme with values ranging from 9–12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved.
PMCID: PMC3364239  PMID: 22666486
8.  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.
PMCID: PMC3517158  PMID: 22396229
fluorescent protein; fluorescence resonance energy transfer (FRET); fluorescence lifetime imaging microscopy (FLIM); acceptor photobleaching; spectral imaging
9.  Mining the Sinorhizobium meliloti Transportome to Develop FRET Biosensors for Sugars, Dicarboxylates and Cyclic Polyols 
PLoS ONE  2012;7(9):e43578.
Förster resonance energy transfer (FRET) biosensors are powerful tools to detect biologically important ligands in real time. Currently FRET bisosensors are available for twenty-two compounds distributed in eight classes of chemicals (two pentoses, two hexoses, two disaccharides, four amino acids, one nucleobase, two nucleotides, six ions and three phytoestrogens). To expand the number of available FRET biosensors we used the induction profile of the Sinorhizobium meliloti transportome to systematically screen for new FRET biosensors.
Methodology/Principal Findings
Two new vectors were developed for cloning genes for solute-binding proteins (SBPs) between those encoding FRET partner fluorescent proteins. In addition to a vector with the widely used cyan and yellow fluorescent protein FRET partners, we developed a vector using orange (mOrange2) and red fluorescent protein (mKate2) FRET partners. From the sixty-nine SBPs tested, seven gave a detectable FRET signal change on binding substrate, resulting in biosensors for D-quinic acid, myo-inositol, L-rhamnose, L-fucose, β-diglucosides (cellobiose and gentiobiose), D-galactose and C4-dicarboxylates (malate, succinate, oxaloacetate and fumarate). To our knowledge, we describe the first two FRET biosensor constructs based on SBPs from Tripartite ATP-independent periplasmic (TRAP) transport systems.
FRET based on orange (mOrange2) and red fluorescent protein (mKate2) partners allows the use of longer wavelength light, enabling deeper penetration of samples at lower energy and increased resolution with reduced back-ground auto-fluorescence. The FRET biosensors described in this paper for four new classes of compounds; (i) cyclic polyols, (ii) L-deoxy sugars, (iii) β-linked disaccharides and (iv) C4-dicarboxylates could be developed to study metabolism in vivo.
PMCID: PMC3454389  PMID: 23028462
10.  Dimerization between aequorea fluorescent proteins does not affect interaction between tagged estrogen receptors in living cells 
Journal of biomedical optics  2008;13(3):031207.
Förster resonance energy transfer (FRET) detection of protein interaction in living cells is commonly measured following the expression of interacting proteins genetically fused to the cyan (CFP) and yellow (YFP) derivatives of the Aequorea victoria fluorescent protein (FP). These FPs can dimerize at mM concentrations, which may introduce artifacts into the measurement of interaction between proteins that are fused with the FPs. Here, FRET analysis of the interaction between estrogen receptors (alpha isoform, ERα) labeled with “wild-type” CFP and YFP is compared with that of ERα labeled with “monomeric” A206K mutants of CFP and YFP. The intracellular equilibrium dissociation constant for the hormone-induced ERα-ERα interaction is similar for ERα labeled with wild-type or monomeric FPs. However, the measurement of energy transfer measured for ERα-ERα interaction in each cell is less consistent with the monomeric FPs. Thus, dimerization of the FPs does not affect the kinetics of ERα-ERα interaction but, when brought close together via ERα-ERα interaction, FP dimerization modestly improves FRET measurement.
PMCID: PMC2581880  PMID: 18601531
fluorescent protein; estrogen receptors; aequorea; Förster resonance energy transfer
11.  Anomalous Surplus Energy Transfer Observed with Multiple FRET Acceptors 
PLoS ONE  2009;4(11):e8031.
Förster resonance energy transfer (FRET) is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (kD→A) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates.
Methodology/Principal Findings
The generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean) and either two or three FRET acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates.
We conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is incorrect.
PMCID: PMC2778011  PMID: 19946626
12.  Lateral Distribution of NBD-PC Fluorescent Lipid Analogs in Membranes Probed by Molecular Dynamics-Assisted Analysis of Förster Resonance Energy Transfer (FRET) and Fluorescence Quenching 
Förster resonance energy transfer (FRET) is a powerful tool used for many problems in membrane biophysics, including characterization of the lateral distribution of lipid components and other species of interest. However, quantitative analysis of FRET data with a topological model requires adequate choices for the values of several input parameters, some of which are difficult to obtain experimentally in an independent manner. For this purpose, atomistic molecular dynamics (MD) simulations can be potentially useful as they provide direct detailed information on transverse probe localization, relative probe orientation, and membrane surface area, all of which are required for analysis of FRET data. This is illustrated here for the FRET pairs involving 1,6-diphenylhexatriene (DPH) as donor and either 1-palmitoyl,2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] hexanoyl)- sn-glycero-3-phosphocholine (C6-NBD-PC) or 1-palmitoyl,2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine (C12-NBD-PC) as acceptors, in fluid vesicles of 1,2-dipalmitoyl-sn-3-glycerophosphocholine (DPPC, 50 °C). Incorporation of results from MD simulations improves the statistical quality of model fitting to the experimental FRET data. Furthermore, the decay of DPH in the presence of moderate amounts of C12-NBD-PC (>0.4 mol%) is consistent with non-random lateral distribution of the latter, at variance with C6-NBD-PC, for which aggregation is ruled out up to 2.5 mol% concentration. These conclusions are supported by analysis of NBD-PC fluorescence self-quenching. Implications regarding the relative utility of these probes in membrane studies are discussed.
PMCID: PMC3509596  PMID: 23203080
DPH; DPPC; fluorescence; FRET; lipid bilayer; membrane probe; molecular dynamics; NBD lipid
13.  Förster Resonance Energy Transfer (FRET) Correlates of Altered Subunit Stoichiometry in Cys-Loop Receptors, Exemplified by Nicotinic α4β2 
We provide a theory for employing Förster resonance energy transfer (FRET) measurements to determine altered heteropentameric ion channel stoichiometries in intracellular compartments of living cells. We simulate FRET within nicotinic receptors (nAChRs) whose α4 and β2 subunits contain acceptor and donor fluorescent protein moieties, respectively, within the cytoplasmic loops. We predict FRET and normalized FRET (NFRET) for the two predominant stoichiometries, (α4)3(β2)2 vs. (α4)2(β2)3. Studying the ratio between FRET or NFRET for the two stoichiometries, minimizes distortions due to various photophysical uncertainties. Within a range of assumptions concerning the distance between fluorophores, deviations from plane pentameric geometry, and other asymmetries, the predicted FRET and NFRET for (α4)3(β2)2 exceeds that of (α4)2(β2)3. The simulations account for published data on transfected Neuro2a cells in which α4β2 stoichiometries were manipulated by varying fluorescent subunit cDNA ratios: NFRET decreased monotonically from (α4)3(β2)2 stoichiometry to mostly (α4)2(β2)3. The simulations also account for previous macroscopic and single-channel observations that pharmacological chaperoning by nicotine and cytisine increase the (α4)2(β2)3 and (α4)3(β2)2 populations, respectively. We also analyze sources of variability. NFRET-based monitoring of changes in subunit stoichiometry can contribute usefully to studies on Cys-loop receptors.
PMCID: PMC3431844  PMID: 22949846
nicotine; cytisine; NFRET; nicotine addiction; Parkinson’s disease; ion channels
14.  Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy 
Nature protocols  2011;6(9):1324-1340.
Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside living cells, including detection of protein-protein interactions. An understanding of the basic physics of fluorescence lifetime measurements is required to use this technique. In this protocol, we describe both the time-correlated single photon counting and the frequency-domain methods for FLIM data acquisition and analysis. We describe calibration of both FLIM systems, and demonstrate how they are used to measure the quenched donor fluorescence lifetime that results from Förster resonance energy transfer (FRET ). We then show how the FLIM-FRET methods are used to detect the dimerization of the transcription factor CCAAT/enhancer binding protein-α in live mouse pituitary cell nuclei. Notably, the factors required for accurate determination and reproducibility of lifetime measurements are described. With either method, the entire protocol including specimen preparation, imaging and data analysis takes ~2 d.
PMCID: PMC3169422  PMID: 21886099
15.  Relative Affinity of Calcium Pump Isoforms for Phospholamban Quantified by Fluorescence Resonance Energy Transfer 
Journal of molecular biology  2010;402(1):210-216.
To investigate regulation of SERCA1a and SERCA2a calcium pump isoforms by phospholamban (PLB), the proteins were fused to fluorescent protein tags and their interactions were quantified by fluorescence resonance energy transfer (FRET) in live cells. For both SERCA1a or SERCA2a, FRET to PLB increased with increasing protein expression level to a maximum value corresponding to a probe separation distance of 64 angstroms. The data indicate the respective regulatory complexes assume the same overall quaternary conformation. However, FRET measurements also revealed that PLB has a 50% higher apparent affinity for SERCA1a relative to SERCA2a. The results suggest that despite structural similarities of the respective regulatory complexes, there is preferential binding of PLB to SERCA1a over SERCA2a. This apparent selectivity may have implications for biochemical studies in which SERCA1a is used as a substitute for SERCA2a. It may also be an important strategic consideration for therapeutic overexpression of SERCA isoforms in cardiac muscle.
PMCID: PMC2935190  PMID: 20643144
phospholamban; SERCA1a; SERCA2a; calcium ATPase; affinity; FRET; calcium handling; membrane proteins
16.  Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET 
Chemistry & biology  2010;17(7):745-755.
We have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or non-fluorescent state (OFF state), respectively. The ON and OFF states exhibit absorbance maxima at 567 and 440 nm, respectively. Due to the photoswitchable absorbance, rsTagRFP can be used as an acceptor for a photochromic Förster resonance energy transfer (pcFRET). The photochromic acceptor facilitates determination of a protein-protein interaction by providing an internal control for FRET. Using pcFRET with EYFP as a donor we observed an interaction between epidermal growth factor receptor and growth factor receptor-binding protein 2 in live cells by detecting the modulation of both the fluorescence intensity and lifetime of the EYFP donor upon the ON-OFF photoswitching of the rsTagRFP acceptor.
PMCID: PMC2911641  PMID: 20659687
17.  Energy migration alters the fluorescence lifetime of Cerulean: implications for fluorescence lifetime imaging Forster resonance energy transfer measurements 
Journal of biomedical optics  2008;13(3):031204.
Förster resonance energy transfer (FRET) is a physical phenomenon used to study molecular interactions in living cells. Changes in the fluorescence lifetime of proteins genetically tagged with a donor fluorophore, such as cyan fluorescent protein or Cerulean, are used to measure energy transfer to a protein tagged with an acceptor fluorophore (yellow fluorescent protein or Venus). Increased transfer efficiency is usually interpreted as closer proximity. Resonance energy transfer is also possible between identical fluorophores. This form of FRET is called energy migration resonance energy transfer (EM-RET). Theoretically, EM-RET should not alter the lifetime or emission spectrum measured from a population of fluorophores. We find a change in the fluorescent lifetime of Cerulean that correlates with energy migration and can result in significant errors when using Cerulean as a donor to measure FRET efficiencies based on fluorescence lifetimes.
PMCID: PMC2556851  PMID: 18601528
energy-migration resonance energy transfer; EM-RET; homo-fret; fluorescent proteins; Cerulean and Venus
18.  Probing Protein Structure in Vivo with FRET 
Fluorescence resonance energy transfer (FRET) is widely used to construct probes for cellular activities and to complement two-hybrid results that predict protein-protein interactions. The Yeast Resource Center promotes an underutilized potential of FRET as an in vivo tool to position proteins within low resolution structures derived from electron microscopy. The success of this approach using widefield microscopy depends upon the choice of filter sets, standardized image acquisition, a robust metric and controls matched to the structure under investigation. A comparison of various CFP and YFP filter combinations from Chroma and Semrock demonstrated the strength of the Chroma filters when coupled with our FRET metric, termed FretR. Coupling CFP and YFP to a selection of proteins of known structure allowed us to create a standard curve of FretR versus distance. How well other FRET metrics conform was also evaluated. Finally FretR was linked to an approximation of the efficiency of energy transfer. Together this feature set has allowed us to contribute to our understanding of the organization of the yeast spindle pole body, cohesin complex and gamma-tubulin complex.
PMCID: PMC3630626
19.  The Impact of Heterogeneity and Dark Acceptor States on FRET: Implications for Using Fluorescent Protein Donors and Acceptors 
PLoS ONE  2012;7(11):e49593.
Förster resonance energy transfer (FRET) microscopy is widely used to study protein interactions in living cells. Typically, spectral variants of the Green Fluorescent Protein (FPs) are incorporated into proteins expressed in cells, and FRET between donor and acceptor FPs is assayed. As appreciable FRET occurs only when donors and acceptors are within 10 nm of each other, the presence of FRET can be indicative of aggregation that may denote association of interacting species. By monitoring the excited-state (fluorescence) decay of the donor in the presence and absence of acceptors, dual-component decay analysis has been used to reveal the fraction of donors that are FRET positive (i.e., in aggregates)._However, control experiments using constructs containing both a donor and an acceptor FP on the same protein repeatedly indicate that a large fraction of these donors are FRET negative, thus rendering the interpretation of dual-component analysis for aggregates between separately donor-containing and acceptor-containing proteins problematic. Using Monte-Carlo simulations and analytical expressions, two possible sources for such anomalous behavior are explored: 1) conformational heterogeneity of the proteins, such that variations in the distance separating donor and acceptor FPs and/or their relative orientations persist on time-scales long in comparison with the excited-state lifetime, and 2) FP dark states.
PMCID: PMC3496711  PMID: 23152925
20.  Fluorescent Fusion Proteins of Soluble Guanylyl Cyclase Indicate Proximity of the Heme Nitric Oxide Domain and Catalytic Domain 
PLoS ONE  2010;5(7):e11617.
To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC) Förster resonance energy transfer (FRET) was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC β1 and α subunits.
Methodology/Principal Findings
Cyan fluorescent protein (CFP) was used as FRET donor and yellow fluorescent protein (YFP) as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged α subunits with the carboxy-terminally tagged β1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the α subunits is close to the carboxy-terminus of the β1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO.
Based on the ability of an amino-terminal fragment of the β1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (αcatβcat), Winger and Marletta (Biochemistry 2005, 44:4083–90) have proposed a direct interaction of the amino-terminal region of β1 with the catalytic domains. In support of such a concept of “trans” regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed “fluorescent-conjoined” sGC's by fusion of the α amino-terminus to the β1 carboxy-terminus leading to a monomeric, fluorescent and functional enzyme complex. To our knowledge this represents the first example where a fluorescent protein links two different subunits of a higher ordered complex to yield a stoichometrically fixed functionally active monomer.
PMCID: PMC2904703  PMID: 20657650
21.  Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions 
Journal of microscopy  2007;228(Pt 2):139-152.
The spectral processed Förster resonance energy transfer (psFRET) imaging method provides an effective and fast method for measuring protein–protein interactions in living specimens. The commercially available linear unmixing algorithms efficiently remove the contribution of donor spectral bleedthrough to the FRET signal. However, the acceptor contribution to spectral bleedthrough in the FRET image cannot be similarly removed, since the acceptor spectrum is identical to the FRET spectrum. Here, we describe the development of a computer algorithm that measures and removes the contaminating ASBT signal in the sFRET image. The new method is characterized in living cells that expressed FRET standards in which the donor and acceptor fluorescent proteins are tethered by amino acid linkers of specific lengths. The method is then used to detect the homo-dimerization of a transcription factor in the nucleus of living cells, and then to measure the interactions of that protein with a second transcription factor.
PMCID: PMC2874973  PMID: 17970914
C/EBPα; confocal; FRET; green fluorescent proteins; protein dimerization; spectral bleedthrough; spectral imaging
22.  Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells 
Molecular Vision  2007;13:854-861.
To determine protein-protein interactions among lens crystallins in living cells.
Fluorescence resonance energy transfer (FRET) microscopy was used to visualize interactions in living cells directly. Two genes, one (αA-crystallin) fused with green fluorescence protein (GFP) and the other (each of the following genes: αB-, βB2-, γC-crystallin, and R120G αB-crystallin mutant) fused with GFP variant red fluorescence protein (RED), were cotransfected into HeLa cells. After culture, confocal microscopy images were taken and FRET values were calculated.
FRET occurs when the two proteins interact. The data show strong interactions between αA- and αB-crystallin and weak interactions between αA- and βB2- or γC-crystallin, which is consistent with our previous two-hybrid system study. The R120G αB-crystallin mutant, however, showed significantly less FRET than wild-type αB-crystallin. There are also more R120G αB-crystallin transfected cells with protein aggregates than wild-type αB-crystallin transfected cells. Cotransfection with αA-crystallin could not rescue R120G αB-crystallin from aggregation.
FRET microscopy gave excellent results on the protein-protein interactions among crystallins. It supports many previous studies and provides a novel technique for further study of protein-protein interactions among lens proteins including membrane and cytoskeletal proteins.
PMCID: PMC2045701  PMID: 17615546
23.  Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells 
Molecular vision  2007;13:854-861.
To determine protein-protein interactions among lens crystallins in living cells.
Fluorescence resonance energy transfer (FRET) microscopy was used to visualize interactions in living cells directly. Two genes, one (αA-crystallin) fused with green fluorescence protein (GFP) and the other (each of the following genes: αB-, βB2-, γC-crystallin, and R120G αB-crystallin mutant) fused with GFP variant red fluorescence protein (RED), were cotransfected into HeLa cells. After culture, confocal microscopy images were taken and FRET values were calculated.
FRET occurs when the two proteins interact. The data show strong interactions between αA- and αB-crystallin and weak interactions between αA- and βB2- or γC-crystallin, which is consistent with our previous two-hybrid system study. The R120G αB-crystallin mutant, however, showed significantly less FRET than wild-type αB-crystallin. There are also more R120G αB-crystallin transfected cells with protein aggregates than wild-type αB-crystallin transfected cells. Cotransfection with αA-crystallin could not rescue R120G αB-crystallin from aggregation.
FRET microscopy gave excellent results on the protein-protein interactions among crystallins. It supports many previous studies and provides a novel technique for further study of protein-protein interactions among lens proteins including membrane and cytoskeletal proteins.
PMCID: PMC2045701  PMID: 17615546
24.  Enhanced Förster Resonance Energy Transfer (FRET) on Single Metal Particle 
We examined the effect of a metallic silver particle on Förster resonance energy transfer (FRET) between a nearby donor-acceptor pair. A donor- labeled oligonucleotide was chemically bound to a single silver particle and then an acceptor- labeled complementary oligonucleotide was conjugated by hybridization. The photophysical behavior of FRET between the donor-acceptor pair on the metal particle was investigated using both ensemble emission spectra and single- molecule fluorescence detections. Both the emission intensities and lifetimes indicated an enhanced FRET efficiency due to the metal particle. This interaction led to an increase in the Förster distance for energy transfer from 8.3 to 13 nm. The rate constant of FRET near the silver particle was 21-fold faster than that of unbound donor-acceptor pair. These results suggest the use of metal-enhanced FRET for measuring proximity of large biomolecules or for energy transfer based assays.
PMCID: PMC2600711  PMID: 19079780
silver nanoparticle; Förster resonance energy transfer (FRET); metal plasmon resonance; metal-enhanced FRET; single molecule fluorescence detection
25.  Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum. 
Nucleic Acids Research  1998;26(21):4946-4952.
The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit.
PMCID: PMC147944  PMID: 9776758

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