Effects on the conformation of FVIIa by sTF and Ca²⁺ binding: studies of fluorescence resonance energy transfer and quenching

The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5? for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4? upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044-1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF. It is known from amidolytic activity measurements that Ca(2+) binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca(2+) binding. Interestingly, this Ca(2+)-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe.     

Synthetic studies of bi-fluorescence-labeled maltooligosaccharides as substrates for α-amylase on the basis of fluorescence resonance energy transfer (FRET)

A series of bi-fluorescence-labeled maltooligosaccharides that lead to fluorescence resonance energy transfer (FRET) was systematically synthesized. Effective FRETs were observed with all of the synthesized probes. Digestion of probes having tetra-, quintet-, hexa- or hepta-saccharidic chain lengths with human saliva α-amylase resulted in disappearance of FRET when an excitation wavelength of at 290nm was used followed by detection at ca. 520nm due to emission from the dansyl moiety. However, continuous FRET was observed when probes having di- or trisaccharidic chain lengths were used as substrates. In addition to the substrate characteristics based on saccharidic chain length, the reaction rates of digestion for the substrates by amylase were different and also depended on their saccharidic chain length.     

Detection of an interaction between prion protein and neuregulin I-β1 by fluorescence resonance energy transfer analysis

Cellular prion protein (PrP) copurifies with neuregulin type I-β1 (NRG I-β1), but no interaction has been detected by a general immunoprecipitation study. We speculate that PrP interacts with NRG I-β1. Here, the interaction of PrP with NRG I-β1 was detected by measuring fluorescence resonance energy transfer (FRET) between enhanced blue (EBFP) and enhanced green (EGFP) fluorescent protein-fusion proteins. Full-length PrP interacted with EGFP in addition to NRG I-β1. From this result, we deduced that PrP interacts with EGFP through its unstructured N-terminal domain. We therefore detected FRET between PrP deleting the N-terminal domain and NRG I-β1. In contrast, the C-terminal domain of PrP interacted with NRG I-β1 and the proteins dissociated completely in the presence of sodium chloride. This interaction occurs at the nanomolar level, which is important for the reaction to be functional in organisms. We concluded that PrP interacted with NRG I-β1 through its C-terminal domain.     

Preimplantation genetic diagnosis of β-thalassemia using real-time polymerase chain reaction with fluorescence resonance energy transfer hybridization probes

Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells (∼5 cells) from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. In the current study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol, permitting molecular genetic analysis to amplify a specific region on the beta-globin (HBB) gene for a couple, carriers of two mutations: c.-78A>G and c.52A>T. Among a total of eight embryos obtained after ovarian stimulation, a single blastomere per embryo at the six- to eight-cell stage was biopsied. This PGD method showed that four embryos were unaffected, two embryos were selected for transfer, and one pregnancy was achieved. Finally, a healthy male baby was delivered at 38 weeks’ gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing.     

Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay

Using the cell-permeable, radioiodinated, irreversible inhibitor BIL-DMK, we probed active cysteine cathepsins in blood. Incubation of the probe in human whole blood followed by separation of white blood cells by dextran sedimentation led to the labeling of one major band at 24 kDa. Two-dimensional gel electrophoresis showed that the band resolved in a single protein spot and corresponded to cathepsin S based on its molecular mass, isoelectric point, and Western blot analysis using anti-human cathepsin S antibodies. Cathepsin S activity in human whole blood was dependent on the time of blood collection, suggesting that cathepsin S activity is subject to circadian variations. Separation of white blood cell populations using a magnetic cell sorter and further characterization by FACS (fluorescent-activated cell sorting) analysis demonstrated that the majority of active cathepsin S resided in the monocyte and neutrophil populations, whereas on a cell basis cathepsin S activity in granulocytes is 10-fold lower than that in monocytes. A whole blood cathepsin S assay was developed and used to measure cathepsin S inhibition in both in vitro and ex vivo conditions. To determine the correlation between the in vitro and ex vivo assays, a reversible cathepsin S inhibitor was dosed intravenously to a rhesus monkey. The inhibitor concentration required to inhibit 50% of the cathepsin S activity ex vivo correlated well with the concentration required to inhibit the enzyme in rhesus monkey whole blood in vitro. The results reported here demonstrate the utility of the activity-based probe BIL-DMK for the ex vivo assessment of cathepsin S inhibition.     

A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E–receptor interactions

The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor, FcεRI, plays a central role in initiating most allergic reactions. The IgE–receptor interaction has been targeted for treatment of allergic diseases, and many high-affinity macromolecular inhibitors have been identified. Small molecule inhibitors would offer significant advantages over current anti-IgE treatment, but no candidate compounds have been identified and fully validated. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR–FRET) assay for monitoring the IgE–receptor interaction. The TR–FRET assay measures an increase in fluorescence intensity as a donor lanthanide fluorophore is recruited into complexes of site-specific Alexa Fluor 488-labeled IgE-Fc and His-tagged FcεRIα proteins. The assay can readily monitor classic competitive inhibitors that bind either IgE-Fc or FcεRIα in equilibrium competition binding experiments. Furthermore, the TR–FRET assay can also be used to follow the kinetics of IgE-Fc–FcεRIα dissociation and identify inhibitory ligands that accelerate the dissociation of preformed complexes, as demonstrated for an engineered DARPin (designed ankyrin repeat protein) inhibitor. The TR–FRET assay is suitable for high-throughput screening (HTS), as shown by performing a pilot screen of the National Institutes of Health (NIH) Clinical Collection Library in a 384-well plate format.     

Use of extremely short F?rster resonance energy transfer probes in real-time polymerase chain reaction

Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed ‘probe-luring’ sequence at their 5′ ends. Hybridization of this sequence to a complementary ‘anchoring’ tail introduced at the 3′ end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a ‘universal’ probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations.     

Use of a novel Förster resonance energy transfer method to identify locations of site-bound metal ions in the U2-U6 snRNA complex

U2 and U6 snRNAs pair to form a phylogenetically conserved complex at the catalytic core of the spliceosome. Interactions with divalent metal ions, particularly Mg(II), at specific sites are essential for its folding and catalytic activity. We used a novel Förster resonance energy transfer (FRET) method between site-bound luminescent lanthanide ions and a covalently attached fluorescent dye, combined with supporting stoichiometric and mutational studies, to determine locations of site-bound Tb(III) within the human U2–U6 complex. At pH 7.2, we detected three metal-ion-binding sites in: (1) the consensus ACACAGA sequence, which forms the internal loop between helices I and III; (2) the four-way junction, which contains the conserved AGC triad; and (3) the internal loop of the U6 intra-molecular stem loop (ISL). Binding at each of these sites is supported by previous phosphorothioate substitution studies and, in the case of the ISL site, by NMR. Binding of Tb(III) at the four-way junction and the ISL sites was found to be pH-dependent, with no ion binding observed below pH 6 and 7, respectively. This pH dependence of metal ion binding suggests that the local environment may play a role in the binding of metal ions, which may impact on splicing activity.     

Detection of protein–protein interactions by a combination of a novel cytoplasmic membrane targeting system of recombinant proteins and fluorescence resonance energy transfer

A novel protein molecular targeting system was created using a cytoplasmic face of a plasma membrane-targeting system in Saccharomyces cerevisiae. The technique involves a molecular display for the creation of a novel reaction site and interaction sites in the field of biotechnology. In a model system, a fluorescent protein was targeted as a reporter to the cytoplasmic face of the plasma membrane. The C-terminal transmembrane domain (CTM) of Ras2p and Snc2p was examined as the portions with anchoring ability to the cytoplasmic face of the plasma membrane. We found that the CTM of Snc2p targeted the enhanced cyan fluorescent protein (ECFP)–protein A fusion protein on the cytoplasmic face of the plasma membrane more strongly than that of Ras2p. To develop it for use as a detection system for protein–protein interactions, the Fc fragment of IgG (Fc) was genetically fused with the enhanced yellow fluorescent protein (EYFP) and expressed in the cytoplasm of the ECFP–protein A-anchored cell. A microscopic analysis showed that fluorescence resonance energy transfer (FRET) between ECFP–protein A and EYFP–Fc occurred, and the change in fluorescence was observed on the cytoplasmic face of the plasma membrane. The detection of protein–protein interactions at the cytoplasmic face of a plasma membrane using FRET combined with a cytoplasmic face-targeting system for proteins provides a novel method for examining the molecular interactions of cytoplasmic proteins, in addition to conventional methods, such as the two-hybrid method in the nuclei. S. Shibasaki and K. Kuroda equally contributed to this work     

What causes the depolarization of trypsin and trypsinogen fluorescence: Intramolecular mobility or non-radiative energy transfer?

Analysis of protein data bank information about the coordinates of definite atoms of protein macromolecules provides an opportunity to evaluate the efficiency of non-radiative resonance energy transfer within the model of fixed, strictly oriented oscillators. Such evaluations for trypsin and trypsinogen (and also for trypsin complex with a pancreatic inhibitor) show that the efficiency of energy transfer among each pair of tryptophan residues is negligibly small. It is also shown that a fairly effective energy transfer from tyrosine to tryptophan residues is possible. The conclusions have been made that the Tyr-Trp energy transfer is one of the factors determining the shape of the trypsin polarization spectrum, and that upon fluorescence excitation at the long-wavelength edge of the absorption spectrum, the depolarization of trypsin fluorescence in aqueous solution at ambient temperature - compared to model compounds (tryptophan, N-acetyltryptophan, glycyltryptophan, etc.), under the conditions of infinite viscosity - is due to the Brownian rotational motion of the macromolecules as a whole as well as the intramolecular mobility. The differences in the level and character of intramolecular mobility of trypsin and trypsinogen are discussed.     

Accurate ab initio calculation of the Ar–CF4 intermolecular potential energy surface

Ar–CF₄ intermolecular interaction potential is studied by ab initio calculations at the MP2 and CCSD(T) levels of theory containing the so-called bond functions ({3s3p2d1f} basis set was chosen) both with and without a correction for the basis-set superposition error. The calculations were performed with Dunning's correlation consistent basis sets (aug-cc-pVXZ, X = D, T, Q, 5) to extrapolate the Ar–CF₄ potential energy minimum and intermolecular distance to their complete basis set (CBS) limits. It is shown that the addition of bond functions results in a dramatic improvement in the convergence of the calculated interaction energies at the MP2/aug-cc-pVTZ level. The MP2/{3s3p2d1f}-aug-cc-pVTZ potential energy surface even approaches the CCSD(T)/aug-cc-pVQZ potential energy surface. The potential energy minima and the intermolecular distances are both significantly closer to the CBS limit when using the bond functions, and it implies that adding bond functions in the calculation has a great effect on the interaction energies. We also find that with bond functions included in the CCSD(T)/aug-cc-pVDZ model chemistry, the potential energy minima are extremely close to the CBS limit and are better than the CCSD(T)/aug-cc-pVQZ values. Several levels of theory described in the text were used to determine pairwise analytic potential energy surfaces for Ar+CF₄. The analytic potential energy surfaces are in very good agreement with the ab initio values.     

Förster resonance energy transfer as a probe of membrane protein folding

The folding reaction of a β-barrel membrane protein, outer membrane protein A (OmpA), is probed with F?rster resonance energy transfer (FRET) experiments. Four mutants of OmpA were generated in which the donor fluorophore, tryptophan, and acceptor molecule, a naphthalene derivative, are placed in various locations on the protein to report the evolution of distances across the bilayer and across the protein pore during a folding event. Analysis of the FRET efficiencies reveals three timescales for tertiary structure changes associated with insertion and folding into a synthetic bilayer. A narrow pore forms during the initial stage of insertion, followed by bilayer traversal. Finally, a long-time component is attributed to equilibration and relaxation, and may involve global changes such as pore expansion and strand extension. These results augment the existing models that describe concerted insertion and folding events, and highlight the ability of FRET to provide insight into the complex mechanisms of membrane protein folding. This article is part of a Special Issue entitled: Membrane protein structure and function.     

satFRET: estimation of Förster resonance energy transfer by acceptor saturation

We demonstrate theoretically and experimentally the quantification of Förster resonance energy transfer (FRET) by direct and systematic saturation of the excited state of acceptor molecules. This version of acceptor depletion methods for FRET estimation, denoted as “satFRET” is reversible and suitable for time-resolved measurements. The technique was investigated theoretically using the steady-state solution of the differential equation system of donor and acceptor molecular states. The influence of acceptor photobleaching during measurement was included in the model. Experimental verification was achieved with the FRET-pair Alexa 546- Alexa 633 loaded on particles in different stoichiometries and measured in a confocal microscope. Estimates of energy transfer efficiency by excited state saturation were compared to those obtained by measurements of sensitised emission and acceptor photobleaching. The results lead to a protocol that allows time-resolved FRET measurements of fixed and living cells on a conventional confocal microscope. This procedure was applied to fixed Chinese hamster ovary cells containing a cyan fluorescent protein and yellow fluorescent protein pair. The time resolution of the technique was demonstrated in a live T cell activation assay comparing the FRET efficiencies measured using a genetically encoded green and red fluorescent protein biosensor for GTP/GDP turnover to those measured by acceptor photobleaching of fixed cells.     

Measurement of internal movements within the 30 S ribosomal subunit using Förster resonance energy transfer

We have used F?rster resonance energy transfer (FRET) to study specific conformational changes in the Escherichia coli 30 S ribosomal subunit that occur upon association with the 50 S subunit. By measuring energy transfer between 13 different pairs of fluorescent probes attached to specific positions on 30 S subunit proteins, we have monitored changes in distance between different locations within the 30 S subunit in its free and 50 S-bound states. The measured distance changes provide restraints for modeling the movement that occurs within the 30 S subunit upon formation of the 70 S ribosome in solution. Treating the head, body, and platform domains of the 30 S subunit as simple rigid bodies, the lowest-energy solution converges on a model that satisfies each of the individual FRET restraints. In this model, the 30 S subunit head tilts towards the 50 S subunit, similar to the movement found in comparing 30 S subunits and 70 S ribosomes from X-ray and cryo-electron microscope structures, and the platform is predicted to undergo a clock-wise rotation upon association.     

Oxazine dye-conjugated dna oligonucleotides: Förster resonance energy transfer in view of molecular dye-DNA interactions

In this work, the photophysical properties of two oxazine dyes (ATTO 610 and ATTO 680) covalently attached via a C6-amino linker to the 5'-end of short single-stranded as well as double-stranded DNA (ssDNA and dsDNA, respectively) of different lengths were investigated. The two oxazine dyes were chosen because of the excellent spectral overlap, the high extinction coefficients, and the high fluorescence quantum yield of ATTO 610, making them an attractive F?rster resonance energy transfer (FRET) pair for bioanalytical applications in the far-red spectral range. To identify possible molecular dye-DNA interactions that cause photophysical alterations, we performed a detailed spectroscopic study, including time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy measurements. As an effect of the DNA conjugation, the absorption and fluorescence maxima of both dyes were bathochromically shifted and the fluorescence decay times were increased. Moreover, the absorption of conjugated ATTO 610 was spectrally broadened, and a dual fluorescence emission was observed. Steric interactions with ssDNA as well as dsDNA were found for both dyes. The dye-DNA interactions were strengthened from ssDNA to dsDNA conjugates, pointing toward interactions with specific dsDNA domains (such as the top of the double helix). Although these interactions partially blocked the dye-linker rotation, a free (unhindered) rotational mobility of at least one dye facilitated the appropriate alignment of the transition dipole moments in doubly labeled ATTO 610/ATTO 680-dsDNA conjugates for the performance of successful FRET. Considering the high linker flexibility for the determination of the donor-acceptor distances, good accordance between theoretical and experimental FRET parameters was obtained. The considerably large F?rster distance of ~7 nm recommends the application of this FRET pair not only for the detection of binding reactions between nucleic acids in living cells but also for monitoring interactions of larger biomolecules such as proteins.     

Correlating calcium binding, Förster resonance energy transfer, and conformational change in the biosensor TN-XXL

Genetically encoded calcium indicators have become instrumental in imaging signaling in complex tissues and neuronal circuits in vivo. Despite their importance, structure-function relationships of these sensors often remain largely uncharacterized due to their artificial and multimodular composition. Here, we describe a combination of protein engineering and kinetic, spectroscopic, and biophysical analysis of the Förster resonance energy transfer (FRET)-based calcium biosensor TN-XXL. Using fluorescence spectroscopy of engineered tyrosines, we show that two of the four calcium binding EF-hands dominate the FRET output of TN-XXL and that local conformational changes of these hands match the kinetics of FRET change. Using small-angle x-ray scattering and NMR spectroscopy, we show that TN-XXL changes from a flexible elongated to a rigid globular shape upon binding calcium, thus resulting in FRET signal output. Furthermore, we compare calcium titrations using fluorescence lifetime spectroscopy with the ratiometric approach and investigate potential non-FRET effects that may affect the fluorophores. Thus, our data characterize the biophysics of TN-XXL in detail and may form a basis for further rational engineering of FRET-based biosensors.     

Quantification of HER expression and dimerization in patients' tumor samples using time-resolved Förster resonance energy transfer

Following the development of targeted therapies against EGFR and HER2, two members of the human epidermal receptor (HER) family of receptor tyrosine kinases, much interest has been focused on their expression in tumors. However, knowing the expression levels of individual receptors may not be sufficient to predict drug response. Here, we describe the development of antibody-based time-resolved Förster resonance energy transfer (TR-FRET) assays for the comprehensive analysis not only of EGFR and HER2 expression in tumor cryosections, but also of their activation through quantification of HER homo- or heterodimers. First, EGFR and HER2 expression levels were quantified in 18 breast tumors and the results were compared with those obtained by using reference methods. The EGFR number per cell determined by TR-FRET was significantly correlated with EGFR mRNA copy number (P<0.0001). Moreover, our method detected HER2 overexpression with 100% specificity and sensibility, as confirmed by the standard IHC, FISH and qPCR analyses. EGFR and HER2 dimerization was then assessed, using as controls xenograft tumors from cell lines with known dimer expression profiles. Our results show that quantification of HER dimerization provides information about receptor activation that cannot be obtained by quantification of single receptors. Quantifying HER expression and dimerization by TR-FRET assays might help identifying novel clinical markers for optimizing patients’ treatment in oncology.     

Elastic deformations of the rotary double motor of single FoF1-ATP synthases detected in real time by Förster resonance energy transfer

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, Förster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8 nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F_OF₁-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F_O motor in the Escherichia coli enzyme is connected to a 3-stepped F₁ motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual F_OF₁-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Single-molecule Förster resonance energy transfer studies of RNA structure, dynamics and function

Single-molecule fluorescence microscopy experiments on RNA molecules brought to light the highly complex dynamics of key biological processes, including RNA folding, catalysis of ribozymes, ligand sensing of riboswitches and aptamers, and protein synthesis in the ribosome. By using highly advanced biophysical spectroscopy techniques in combination with sophisticated biochemical synthesis approaches, molecular dynamics of individual RNA molecules can be observed in real time and under physiological conditions in unprecedented detail that cannot be achieved with bulk experiments. Here, we review recent advances in RNA folding and functional studies of RNA and RNA-protein complexes addressed by using single-molecule Förster (fluorescence) resonance energy transfer (smFRET) technique.