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PubMed2005

Measurements of internal distance changes of the 30S ribosome using FRET with multiple donor-acceptor pairs: quantitative spectroscopic methods.

We present analytical and experimental procedures for determining distance changes within the 30 S subunit of the Escherichia coli ribosome using Förster resonance energy transfer (FRET). We discuss ways to contend with complexities when using FRET to measure distance changes within large multi-subunit macromolecular complexes, such as the ribosome. Complications can arise due to non-stoichiometric labeling of donor and acceptor probes, as well as environmental effects that are specific to each conjugation site. We show how to account for changes in extinction coefficients, quenching, labeling stoichiometry and other variations in the spectroscopic properties of the dye to enable more accurate calculation of distances from FRET data. We also discuss approximations that concern the orientation of the transition moments of the two dye molecules, as well as the impact of other errors in the measurement of absolute distances. Thirteen dye-pair locations with different distances using 18 independent FRET pairs conjugated to specific 30 S protein residues have been used to determine distance changes within the 30 S subunit upon association with the 50 S subunit, forming the 70 S ribosome. Here, we explain the spectroscopic methods we have used, which should be of general interest in studies that aim at obtaining quantitative distance information from FRET.

Crystallography, X-Ray↗
PubMed2005

Improvement of a FRET-based indicator for cAMP by linker design and stabilization of donor-acceptor interaction.

Förster resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for a variety of intracellular molecular events. Often, however, the poor dynamic range of such reporters prevents detection of subtle but physiologically relevant signals. Here we present a strategy for improving FRET efficiency between donor and acceptor fluorophores in a green fluorescent protein (GFP)-based protein indicator for cAMP. Such indicator is based on protein kinase A (PKA) and was generated by fusion of CFP and YFP to the regulatory and catalytic subunits of PKA, respectively. Our approach to improve FRET efficiency was to perform molecular dynamic simulations and modelling studies of the linker peptide (L11) joining the CFP moiety and the regulatory subunit in order to define its structure and use this information to design an improved linker. We found that L11 contains the X-Y-P-Y-D motif, which adopts a turn-like conformation that is stiffly conserved along the simulation time. Based on this finding, we designed a new linker, L22 in which the YPY motif was doubled in order to generate a stiffer peptide and reduce the mobility of the chromophore within the protein complex, thus favouring CFP/YFP dipole-dipole interaction and improving FRET efficiency. Molecular dynamic simulations of L22 showed, unexpectedly, that the conformational behaviour of L22 was very loose. Based on the analysis of the three principal conformational states visited by L22 during the simulation time, we modified its sequence in order to increase its rigidity. The resulting linker L20 displayed lower flexibility and higher helical content than L22. When inserted in the cAMP indicator, L20 yielded a probe showing almost doubled FRET efficiency and a substantially improved dynamic range.

Amino Acid Sequence↗
PubMed2006

Single-molecule FRET study of denaturant induced unfolding of RNase H.

Single-molecule fluorescence (Förster) resonance energy transfer (FRET) experiments were performed on surface-immobilized RNase H molecules as a function of the concentration of the chemical denaturant guanidinium chloride (GdmCl). For comparison, we measured ensemble FRET on RNase H solutions. The single-molecule approach allowed us to study FRET distributions of the subpopulation of unfolded molecules without interference from the folded population. The unfolded ensemble experienced a continuous shift of the FRET efficiency distribution with increasing concentration of GdmCl, indicating a heterogeneous population of expanding, unfolded polypeptide chains. We have analyzed the behavior of the unfolded state quantitatively with a model in which the unfolded state is described by a continuum of substates, with the free energy of each substate linearly coupled to its m-value, the proportionality coefficient between free energy and denaturant activity. By fitting this model to the data, we have derived energetic and structural parameters that describe the unfolded state ensemble. Specifically, we have found that the average size of the unfolded state increases from 23-38 A between 0 and 6 M denaturant. Excellent agreement was achieved between the fitted model and our FRET measurements, and with previously published nuclear magnetic resonance and small-angle X-ray scattering data.

Data Interpretation, Statistical↗
PubMed2005

Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes.

GFP-based fluorescence resonance energy transfer (FRET) probes that visualize local activity-changes of Ras and Rho GTPases in living cells are now available for examining the spatio-temporal regulation of these proteins. This article describes principles and strategies to develop intramolecular FRET probes for Ras- and Rho-family GTPases. The procedure for characterizing candidate probes, and image acquisition and processing are also explained. An optimal FRET probe should have (i) a wide dynamic range (which means a high sensitivity), (ii) a high fluorescence intensity, (iii) target specificity, and (iv) a minimal perturbation to endogenous signaling cascades. Although an improvement of FRET probes should be executed in a trial-and-error manner, practical tips for optimization are provided here. In addition, we illustrate some applications of FRET probes for neuronal cells, which are composed of diverse subcellular compartments with different functions; thus, tools to decipher the dynamics of GTPase activity in each compartment have long been desired.

Animals↗
PubMed2005

Visualization of Rab5 activity in living cells using FRET microscopy.

Rab5 is a member of the large family of small GTPases involved in membrane trafficking. Two genetically encoded sensors were developed to visualize Rab5 in its GTP-bound conformation in living cells. Rab5-binding fragments of Rabaptin5 or early endosomal antigen 1 (EEA.1) were fused to yellow fluorescent protein (YFP) and used in the fluorescent resonance energy transfer (FRET) assay together with Rab5-tagged cyan fluorescent protein (CFP). The presence of energy transfer between CFP-Rab5 and YFP-Rab5 binding fragments detected by sensitized FRET microscopy has validated the utility of these generated sensors to visualize the localization of GTP-bound Rab5. GTP-bound Rab5 was found in endosomes, often concentrated in distinct microdomains. Molecular architecture of the Rab5 microdomains was analyzed by three-chromophore FRET (3-FRET) microscopy, utilizing YFP, CFP, and monomeric red fluorescent proteins (mRFP.l). The results of the 3-FRET analysis suggest that GTP-bound Rab5 is capable of oligomerization and present in multiprotein complexes.

Animals↗
PubMed2003

Development of a novel FRET immunosensor technique.

We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.

Antigen-Antibody Complex↗
PubMed2003

Fluorescence resonance energy transfer (FRET) as a high-throughput assay for coupling reactions. Arylation of amines as a case study.

A solution-phase assay based on fluorescence resonance energy transfer (FRET) was developed for high-throughput screening of palladium catalyzed aminations of aryl halides. Dansylpiperazine was used as the fluorescent component and a chloro- or bromoarene tagged with an azodye as the quenching partner. Fluorescence intensities of reaction aliquots correlated linearly with reaction yield after dilution to appropriate concentrations. A library of 119 phosphine and heterocyclic carbene ligands was evaluated in duplicate reactions of two combinations. In general, the FRET assay displayed excellent reproducibility, with less than 5% of the duplicate experiments showing significant variability in yields. Among reactions producing greater than 50% yield, the average percent uncertainty was just 5%. For a small subset of sterically hindered ligands, differences in yields between 10 and 20% were observed between the substrates bearing dyes for the FRET assay and substrates that are unfunctionalized. However, the remaining catalyst combinations gave yields similar to those expected from literature precedent. In addition to an evaluation of the accuracy of the FRET assay, this work includes the use of the FRET assay to investigate relative activities of various catalysts for the amination of aryl bromides and chlorides and to find conditions for aminations in more polar solvents. Reactions with K(3)PO(4) base in aqueous mixtures of polar and nonpolar organic solvents were shown to be appropriate for the amination chemistry.

Amines↗
PubMed2001

Imaging FRET between spectrally similar GFP molecules in single cells.

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

Animals↗
PubMed2005

Evolutionary optimization of fluorescent proteins for intracellular FRET.

Fluorescent proteins that exhibit Forster resonance energy transfer (FRET) have made a strong impact as they enable measurement of molecular-scale distances through changes in fluorescence. FRET-based approaches have enabled otherwise intractable measurements of molecular concentrations, binding interactions and catalytic activity, but are limited by the dynamic range and sensitivity of the donor-acceptor pair. To address this problem, we applied a quantitative evolutionary strategy using fluorescence-activated cell sorting to optimize a cyan-yellow fluorescent protein pair for FRET. The resulting pair, CyPet-YPet, exhibited a 20-fold ratiometric FRET signal change, as compared to threefold for the parental pair. The optimized FRET pair enabled high-throughput flow cytometric screening of cells undergoing caspase-3-dependent apoptosis. The CyPet-YPet energy transfer pair provides substantially improved sensitivity and dynamic range for a broad range of molecular imaging and screening applications.

Bacterial Proteins↗
PubMed2006

FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells.

The lateral organization of a prototypical G protein-coupled receptor, the neurokinin-1 receptor (NK1R), was investigated in living cells by fluorescence resonance energy transfer (FRET) microscopy, taking advantage of the recently developed acyl carrier protein (ACP) labeling technique. The NK1R was expressed as fusion protein with ACP to which small fluorophores were then covalently bound. Our approach allowed the recording of FRET images of receptors on living cells with unprecedented high signal-to-noise ratios and a subsequent unequivocal quantification of the FRET data owing to (i) the free choice of optimal fluorophores, (ii) the labeling of NK1Rs exclusively on the cell surface, and (iii) the precise control of the donor-acceptor molar ratio. Our single-cell FRET measurements exclude the presence of constitutive or ligand-induced homodimers or oligomers of NK1Rs. The strong dependence of FRET on the receptor concentration further reveals that NK1Rs tend to concentrate in microdomains, which are found to constitute approximately 1% of the cell membrane and to be sensitive to cholesterol depletion.

Acyl Carrier Protein↗
PubMed2006

Intracellular protein interaction mapping with FRET hybrids.

A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using FRET between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types.

Cell Line↗
PubMed2005

Determination of tumor necrosis factor receptor-associated factor trimerization in living cells by CFP->YFP->mRFP FRET detected by flow cytometry.

The availability of protein fluorophores with appropriate spectral properties has made it possible to employ fluorescence resonance energy transfer (FRET) to assess interactions between three proteins microscopically. Flow cytometry offers excellent sensitivity, effective signal separation and the capacity to assess a large number of events, and, therefore, should be an ideal means to explore protein interactions in living cells. Here, we report a flow-cytometric FRET technique that employed both direct energy transfer from CFP-->YFP-->mRFP and donor quenching to assess TRAF2 trimerization in living cells. Initially, a series of fusion proteins incorporating CFP, YFP and mRFP with spacers that did or did not permit FRET were employed to document the magnitude of CFP-->YFP and YFP-->mRFP FRET and to calculate the efficiency of CFP-->YFP-->mRFP two-step FRET. Based upon this, TRAF2 homotrimerization could be detected. This method should have great utility in studying the dynamics of interactions between three specific proteins in vivo.

Bacterial Proteins↗
PubMed2004

Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells.

We present a novel, multi-dimensional, time-correlated single photon counting (TCSPC) technique to perform fluorescence lifetime imaging with a laser-scanning microscope operated at a pixel dwell-time in the microsecond range. The unsurpassed temporal accuracy of this approach combined with a high detection efficiency was applied to measure the fluorescent lifetimes of enhanced cyan fluorescent protein (ECFP) in isolation and in tandem with EYFP (enhanced yellow fluorescent protein). This technique enables multi-exponential decay analysis in a scanning microscope with high intrinsic time resolution, accuracy and counting efficiency, particularly at the low excitation levels required to maintain cell viability and avoid photobleaching. Using a construct encoding the two fluorescent proteins separated by a fixed-distance amino acid spacer, we were able to measure the fluorescence resonance energy transfer (FRET) efficiency determined by the interchromophore distance. These data revealed that ECFP exhibits complex exponential fluorescence decays under both FRET and non-FRET conditions, as previously reported. Two approaches to calculate the distance between donor and acceptor from the lifetime delivered values within a 10% error range. To confirm that this method can be used also to quantify intermolecular FRET, we labelled cultured neurones with the styryl dye FM1-43, quantified the fluorescence lifetime, then quenched its fluorescence using FM4-64, an efficient energy acceptor for FM1-43 emission. These experiments confirmed directly for the first time that FRET occurs between these two chromophores, characterized the lifetimes of these probes, determined the interchromophore distance in the plasma membrane and provided high-resolution two-dimensional images of lifetime distributions in living neurones.

Animals↗
PubMed2005

Simulation of fretting wear at orthopaedic implant interfaces.

Osteolysis due to wear debris is a primary cause of failure of total joint replacements. Although debris produced by the joint articulating surfaces has been studied and simulated extensively, fretting wear debris, produced at nonarticulating surfaces, has not received adequate attention. We developed a three-station fretting wear simulator to reproduce in vivo motion and stresses at the interfaces of total joint replacements. The simulator is based on the beam bending theory and is capable of producing cyclic displacement from 3 to 1000 microns, under varying magnitudes of contact stresses. The simulator offers three potential advantages over previous studies: The ability to control the displacement by load, the ability to produce very small displacements, and dynamic normal loads as opposed to static. A pilot study was designed to test the functionality of the simulator, and verify that calculated displacements and loads produced the predicted differences between two commonly used porous ingrowth titanium alloy surfaces fretting against cortical bone. After 1.5 million cycles, the simulator functioned as designed, producing greater wear of bone against the rougher plasma-sprayed surface compared to the fiber-mesh surface, as predicted. A novel pin-on-disk apparatus for simulating fretting wear at orthopaedic implant interfaces due to micromotion is introduced. The test parameters measured with the fretting wear simulator were as predicted by design calculations, and were sufficient to measure differences in the height and weight of cortical bone pins rubbing against two porous ingrowth surfaces, plasma-sprayed titanium and titanium fiber mesh.

Cadaver↗
PubMed2004

Conformational changes of the Ca(2+) regulatory site of the Na(+)-Ca(2+) exchanger detected by FRET.

The Na(+)-Ca(2+) exchanger is a plasma membrane protein expressed at high levels in cardiomyocytes. It extrudes 1 Ca(2+) for 3 Na(+) ions entering the cell, regulating intracellular Ca(2+) levels and thereby contractility. Na(+)-Ca(2+) exchanger activity is regulated by intracellular Ca(2+), which binds to a region (amino acids 371-508) within the large cytoplasmic loop between transmembrane segments 5 and 6. Regulatory Ca(2+) activates the exchanger and removes Na(+)-dependent inactivation. The physiological role of intracellular Ca(2+) regulation of the exchanger is not yet established. Yellow (YFP) and cyan (CFP) fluorescent proteins were linked to the NH(2)- and CO(2)H-termini of the exchanger Ca(2+) binding domain (CBD) to generate a construct (YFP-CBD-CFP) capable of responding to changes in intracellular Ca(2+) concentrations by FRET efficiency measurements. The two fluorophores linked to the CBD are sufficiently close to generate FRET. FRET efficiency was reduced with increasing Ca(2+) concentrations. Titrations of Ca(2+) concentration versus FRET efficiency indicate a K(D) for Ca(2+) of approximately 140 nM, which increased to approximately 400 nM in the presence of 1 mM Mg(2+). Expression of YFP-CBD-CFP in myocytes, generated changes in FRET associated with contraction, suggesting that NCX is regulated by Ca(2+) on a beat-to-beat basis during excitation-contraction coupling.

Animals↗
PubMed2004

Distance-restrained docking of rifampicin and rifamycin SV to RNA polymerase using systematic FRET measurements: developing benchmarks of model quality and reliability.

We are developing distance-restrained docking strategies for modeling macromolecular complexes that combine available high-resolution structures of the components and intercomponent distance restraints derived from systematic fluorescence resonance energy transfer (FRET) measurements. In this article, we consider the problem of docking small-molecule ligands within macromolecular complexes. Using simulated FRET data, we have generated a series of benchmarks that permit estimation of model accuracy based on the quantity and quality of FRET-derived distance restraints, including the number, random error, systematic error, distance distribution, and radial distribution of FRET-derived distance restraints. We find that expected model accuracy is 10 A or better for models based on: i), > or =20 restraints with up to 15% random error and no systematic error, or ii), > or =20 restraints with up to 15% random error, up to 10% systematic error, and a symmetric radial distribution of restraints. Model accuracies can be improved to 5 A or better by increasing the number of restraints to > or =40 and/or by optimizing the distance distribution of restraints. Using experimental FRET data, we have defined the positions of the binding sites within bacterial RNA polymerase of the small-molecule inhibitors rifampicin (Rif) and rifamycin SV (Rif SV). The inferred binding sites for Rif and Rif SV were located with accuracies of, respectively, 7 and 10 A relative to the crystallographically defined binding site for Rif. These accuracies agree with expectations from the benchmark simulations and suffice to indicate that the binding sites for Rif and Rif SV are located within the RNA polymerase active-center cleft, overlapping the binding site for the RNA-DNA hybrid.

Binding Sites↗
PubMed2004

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

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

Animals↗
PubMed2006

Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells.

Measurement of fluorescence resonance energy transfer (FRET) efficiency and the relative concentration of donor and acceptor fluorophores in living cells using the three-filter cube approach requires the determination of two constants: 1), the ratio of sensitized acceptor emission to donor fluorescence quenching (G factor) and 2), the ratio of donor/acceptor fluorescence intensity for equimolar concentrations in the absence of FRET (k factor). We have developed a method to determine G and k that utilizes two donor-acceptor fusion proteins with differing FRET efficiencies-the value of which need not be known. We validated the method by measuring the FRET efficiency and concentration ratio of the fluorescent proteins Cerulean and Venus in mammalian cells expressing a series of fusion proteins with varying stoichiometries. The method greatly simplifies quantitative FRET measurement in living cells as it does not require cell fixation, acceptor photobleaching, protein purification, or specialized equipment for determining fluorescence spectra or lifetime.

Algorithms↗