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PubMed1998

Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

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.

Animals↗
PubMed2004

A new approach to SNP genotyping with fluorescently labeled mononucleotides.

Fluorescence resonance energy transfer (FRET) is one of the most powerful and promising tools for single nucleotide polymorphism (SNP) genotyping. However, the present methods using FRET require expensive reagents such as fluorescently labeled oligonucleotides. Here, we describe a novel and cost-effective method for SNP genotyping using FRET. The technique is based on allele-specific primer extension using mononucleotides labeled with a green dye and a red dye. When the target DNA contains the sequence complementary to the primer, extension of the primer incorporates the green and red dye-labeled nucleotides into the strand, and red fluorescence is emitted by FRET. In contrast, when the 3' end nucleotide of the primer is not complementary to the target DNA, there is no extension of the primer, or FRET signal. Therefore, discrimination among genotypes is achieved by measuring the intensity of red fluorescence after the extension reaction. We have validated this method with 11 SNPs, which were successfully determined by end-point measurements of fluorescence intensity. The new strategy is simple and cost-effective, because all steps of the preparation consist of simple additions of solutions and incubation, and the dye-labeled mononucleotides are applicable to all SNP analyses. This method will be suitable for large-scale genotyping.

Alleles↗
PubMed2002

Ca2+- and S1-induced conformational changes of reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy: structural evidence for three States of thin filament.

Rabbit skeletal muscle alpha-tropomyosin (Tm) and the deletion mutant (D234Tm) in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056] were used to investigate the interaction between actin and tropomyosin or actin and troponin (Tn) by means of fluorescence resonance energy transfer (FRET). FRET between Cys-190 of D234Tm and Gln-41 or Cys-374 of actin did not cause any significant Ca2+-induced movement of D234Tm, as reported previously for native Tm [Miki et al. (1998) J. Biochem. 123, 1104-1111]. FRET did not show any significant S1-induced movement of Tm and D234Tm on thin filaments either. The distances between Cys-133 of TnI, and Gln-41 and Cys-374 of actin on thin filaments reconstituted with D234Tm (mutant thin filaments) were almost the same as those on thin filaments with native Tm (wild-type thin filaments) in the absence of Ca2+. Upon binding of Ca2+ to TnC, these distances on mutant thin filaments increased by approximately 10 A in the same way as on wild-type thin filaments, which corresponds to a Ca2+-induced conformational change of thin filaments [Miki et al. (1998) J. Biochem. 123, 324-331]. The rigor binding of myosin subfragment 1 (S1) further increased these distances by approximately 7 A on both wild-type and mutant thin filaments when the thin filaments were fully decorated with S1. This indicates that a further conformational change on thin filaments was induced by S1 rigor-binding (S1-induced or open state). Plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed that the curve for wild-type thin filaments is hyperbolic, whereas that for mutant thin filaments is sigmoidal. This suggests that the transition to the S1-induced state on mutant thin filaments is depressed with a low population of rigor S1. In the absence of Ca2+, the distance also increased on both wild-type and mutant thin filaments close to the level in the presence of Ca2+ as the molar ratio of S1 to actin increased up to 1. The curves are sigmoidal for both wild-type and mutant thin filaments. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding. For mutant thin filaments, the transition from the closed state to the open state in the presence of ATP is strongly depressed, which results in the inhibition of acto-myosin ATPase even in the presence of Ca2+. The present FRET measurements provide structural evidence for three states of thin filaments (relaxed, Ca2+-induced or closed, and S1-induced or open states) for the regulation mechanism of skeletal muscle contraction.

Actin Cytoskeleton↗
PubMed2006

Computational modeling of a new fluorescent biosensor for caspase proteolytic activity improves dynamic range.

The class of fluorescence resonance energy transfer (FRET) protein biosensors that are useful for measuring protease activity is composed of a tandem fusion of yellow fluorescent protein (YFP), a cleavage recognition sequence, and cyan fluorescent protein (CFP). The dynamic range of these FRET-based protein biosensors is often weak, but applications such as high throughput drug screening require stronger dynamic ranges. Using the biosensor for the caspase-3 protease as an example, here we showed a computational approach to improve the FRET dynamic range based on the atomic structure of caspase-3 bound to its inhibitor. This result was verified from our experiments where the FRET dynamic range improved by at least 60% on average in both in vitro and in vivo contexts. In concept, the same strategy can be applied to improve dynamic range of other FRET-based protein biosensors for protease activity where there exist solved atomic structures for protein complexes.

Caspase 3↗
PubMed2005

Development of a real-time fluorescence resonance energy transfer PCR to identify the main pathogenic Campylobacter spp.

A simple real-time fluorescence resonance energy transfer (FRET) PCR, targeting the gyrA gene outside the quinolone resistance-determining region, was developed to identify Campylobacter jejuni and Campylobacter coli. These species were distinguished easily, as the corresponding melting points showed a difference of 15 degrees C. A second assay using the same biprobe and PCR conditions, but different PCR primers, was also developed to identify the less frequently encountered Campylobacter fetus. These assays were applied to 807 Campylobacter isolates from clinical specimens. Compared to phenotypic identification tests, the FRET assay yielded the same results for all except three of the isolates. Analysis by standard PCR and 16S rDNA sequencing demonstrated that two of these isolates were hippurate-negative C. jejuni strains, resulting in an erroneous phenotypic identification, while the third was an isolate of C. coli that contained a gyrA gene typical of C. jejuni, resulting in misidentification by the FRET assay. The FRET assay identified more isolates than standard PCR, which failed to yield amplification products with c. 10% of isolates. It was concluded that the FRET assays were rapid, reliable, reproducible and relatively cost-efficient, as they require only one biprobe and can be performed directly on boiled isolates.

Amino Acid Sequence↗
PubMed2001

Fluorescence resonance energy transfer microscopy: a mini review.

Fluorescence resonance energy transfer (FRET) microscopy is a better method than the x-ray diffraction, nuclear magnetic resonance, or electron microscopy for studying the structure and localization of proteins under physiological conditions. In this paper, we describe four different light microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBPalpha) in living pituitary cells. In wide-field, confocal, and two-photon microscopy the FRET image provides two-dimensional spatial distribution of steady-state protein-protein interactions. The two-photon imaging technique provides a better FRET signal (less bleedthrough and photobleaching) compared to the other two techniques. This information, although valuable, falls short of revealing transient interactions of proteins in real time. The fluorescence lifetime methods allow us to monitor FRET signals at the moment of the protein interactions at a resolution on the order of subnanoseconds, providing high temporal, as well as spatial resolution. This paper will provide a brief review of the above-mentioned FRET techniques.

CCAAT-Enhancer-Binding Proteins↗
PubMed2003

Spectroscopic approach for monitoring two-photon excited fluorescence resonance energy transfer from homodimers at the subcellular level.

We have employed a spectroscopic approach for monitoring fluorescence resonance energy transfer (FRET) in living cells. This method provides excellent spectral separation of green fluorescent protein (GFP) mutant signals within a subcellular imaging volume using two-photon excited fluorescence imaging and spectroscopy (TPIS-FRET). In contrast to current FRET-based methodologies, TPIS-FRET does not rely on the selection of optical filters, ratiometric image analysis, or bleedthrough correction algorithms. Utilizing the intrinsic optical sectioning capabilities of TPIS-FRET, we have identified protein-protein interactions within discrete subcellular domains. To illustrate the applicability of this technique to the detection of homodimer formation, we demonstrated the in vivo association of promyleocyte (PML) homodimers within their corresponding nuclear body.

Cell Line, Tumor↗
PubMed2003

Use of the Roche LightCycler instrument in a real-time PCR for Trichomonas vaginalis in urine samples from females and males.

Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted infection (STI) that can result in vaginitis, urethritis, and preterm birth. Traditional methods of diagnosis, including wet preparation, can be unreliable. In this study, we describe the adaptation of an existing PCR method for specific detection of T. vaginalis DNA into a rapid real-time PCR assay based on fluorescence resonance energy transfer (FRET) probe chemistry. The FRET-based assay described demonstrated high sensitivity with a detection limit of 1.06 organisms, as well as a high specificity. A total of 253 urine samples collected prospectively from both men and women were tested for T. vaginalis DNA with both the FRET-based assay and a previously validated PCR assay. When the validated PCR assay was used as the "gold standard" and after discrepancies had been resolved, our FRET-based assay demonstrated an analytical sensitivity and specificity of 90.1 and 100%, respectively. Overall results suggest that FRET-based assays can provide rapid, accurate, and high-throughput detection of T. vaginalis and may prove useful in clinical settings and for large-scale screening programs.

Animals↗
PubMed2003

Spatial distribution analysis of AT- and GC-rich regions in nuclei using corrected fluorescence resonance energy transfer.

We employed microscopic intensity-based fluorescence resonance energy transfer (FRET) images with correction by donor and acceptor concentrations to obtain unbiased maps of spatial distribution of the AT- and GC-rich DNA regions in nuclei. FRET images of 137 bovine aortic endothelial cells stained by the AT-specific donor Hoechst 33258 and the GC-specific acceptor 7-aminoactinomycin D were acquired and corrected for the donor and acceptor concentrations by the Gordon's method based on the three fluorescence filter sets. The corrected FRET images were quantitatively analyzed by texture analysis to correlate the spatial distribution of the AT- and GC-rich DNA regions with different phases of the cell cycle. Both visual observation and quantitative texture analysis revealed an increased number and size of the low FRET efficiency centers for cells in the G(2)/M-phases, compared to the G(1)-phase cells. We have detected cell cycle-dependent changes of the spatial organization and separation of the AT- and GC-rich DNA regions. Using the corrected FRET (cFRET) technique, we were able to detect early DNA separation stages in late interphase nuclei.

AT Rich Sequence↗
PubMed1998

Nuclear receptors have distinct affinities for coactivators: characterization by fluorescence resonance energy transfer.

UNLABELLED: Ligand-dependent interactions between nuclear receptors and members of a family of nuclear receptor coactivators are associated with transcriptional activation. Here we used fluorescence resonance energy transfer (FRET) as an approach for detecting and quantitating such interactions. Using the ligand binding domain (LBD) of peroxisome proliferator-activated receptor (PPARgamma) as a model, known agonists (thiazolidinediones and delta12, 14-PGJ2) induced a specific interaction resulting in FRET between the fluorescently labeled LBD and fluorescently labeled coactivators [CREB-binding protein (CBP) or steroid receptor coactivator-1 (SRC-1)]. Specific energy transfer was dose dependent; individual ligands displayed distinct potency and maximal FRET profiles that were identical when results obtained using CBP vs. SRC-1 were compared. In addition, half-maximally effective agonist concentrations (EC59s) correlated well with reported results using cell-based assays. A site-directed AF2 mutant of PPARgamma (E471A) that abrogated ligand-stimulated transcription in transfected cells also failed to induce ligand-mediated FRET between PPARgamma LBD and CBP or SRC-1. Using estrogen receptor (ERalpha) as an alternative system, known agonists induced an interaction between ERalpha LBD and SRC-1, whereas ER antagonists disrupted agonist-induced interaction of ERalpha with SRC-1. In the presence of saturating agonist concentrations, unlabeled CBP or SRC-1 was used to compete with fluorescently labeled coactivators with saturation kinetics. Relative affinities for the individual receptor-coactivator pairs were determined as follows: PPARgamma-CBP = ERalpha-SRC-1 > PPARgamma-SRC-1 >> ERalpha-CBP. CONCLUSIONS: 1) FRET-based coactivator association is a novel approach for characterizing nuclear receptor agonists or antagonists; individual ligands display potencies that are predictive of in vivo effects and distinct profiles of maximal activity that are suggestive of alternative receptor conformations. 2) PPARgamma interacts with both CBP and SRC-1; transcriptional activation and coactivator association are AF2 dependent. 3) Nuclear receptor LBDs have distinct affinities for individual coactivators; thus, PPARgamma has a greater apparent affinity for CBP than for SRC-1, whereas ERalpha interacts preferentially with SRC-1 but very weakly with CBP.

Animals↗
PubMed2005

Simultaneous real-time detection of initiator- and effector-caspase activation by double fluorescence resonance energy transfer analysis.

Fluorescence resonance energy transfer (FRET) with green fluorescent protein (GFP) variants has become widely used for biochemical research. In order to expand the choice of fluorescent range in FRET analysis, we designed various color versions of the FRET-based probes for caspase activity, in which the substrate sequence of the caspase was sandwiched by donor and acceptor fluorescent proteins, and studied the potential of these color versions as fluorescent indicators. Six color versions were constructed by a combination of cyan fluorescent protein (CFP), GFP, yellow fluorescent protein (YFP), and DsRed. Real-time monitoring in single cells revealed that all probes could detect caspase activation during tumor necrosis factor (TNF)-alpha-induced cell death as a fluorescent change. GFP-DsRed and YFP-DsRed were as sensitive as CFP-YFP, and CFP-DsRed also showed a large fluorescent change. By using two probes, CFP-DsRed and YFP-DsRed, we carried out simultaneous multi-FRET analysis and revealed that the initiator- and effector-caspases were activated almost simultaneously in TNF-alpha-induced cell death. These findings may give experimental bases for the development of novel techniques to analyze multi-events simultaneously in single cells by using FRET probes in combination.

Apoptosis↗
PubMed2006

Detecting and imaging protein-protein interactions during G protein-mediated signal transduction in vivo and in situ by using fluorescence-based techniques.

An important goal in cell biology has been to observe dynamic interactions between protein molecules within a living cell as they execute the reactions of a particular biochemical pathway. An important step toward achieving this goal has been the development of noninvasive fluorescence-based detection and imaging techniques for determining whether and when specific biomolecules in a cell become associated with one another. Furthermore, these techniques, which take advantage of phenomena known as bioluminescence- and fluorescence resonance energy transfer (BRET and FRET, respectively) as well as bimolecular fluorescence complementation (BiFC), can provide information about where and when protein-protein interactions occur in the cell. Increasingly BRET, FRET, and BiFC are being used to probe interactions between components involved in G protein- mediated signal transduction. Heptahelical (7TM) receptors, heterotrimeric guanine nucleotide binding proteins (G proteins) and their proximal downstream effectors constitute the core components of these ubiquitous signaling pathways. Signal transduction is initiated by the binding of agonist to heptahelical (7TM) receptors that in turn activate their cognate G proteins. The activated G protein subsequently regulates the activity of specific effectors. 7TM receptors, G proteins, and effectors are all membrane-associated proteins, and for decades two opposing hypotheses have vied for acceptance. The predominant hypothesis has been that these proteins move about independently of one another in membranes and that signal transduction occurs when they encounter each other as the result of random collisions. The contending hypothesis is that signaling is propagated by organized complexes of these proteins. Until recently, the data supporting these hypotheses came from studying signaling proteins in solution, in isolated membranes, or in fixed cells. Although the former hypothesis has been favored, recent studies using BRET and FRET have generally supported the latter hypothesis as being the most likely scenario operating in living cells. In addition to the core components, there are many other proteins involved in G protein signaling, and BRET and FRET studies have been used to investigate their interactions as well. This review describes various BRET, FRET, and BiFC techniques, how they have been or can be applied to the study of G protein signaling, what caveats are involved in interpreting the results, and what has been learned about G protein signaling from the published studies.

Cell Physiological Phenomena↗
PubMed2005

Single-molecule studies of synaptotagmin and complexin binding to the SNARE complex.

The assembly of multiprotein complexes at the membrane interface governs many signaling processes in cells. However, very few methods exist for obtaining biophysical information about protein complex formation at the membrane. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in deposited lipid bilayers. Using total internal reflectance microscopy, individual binding events at the membrane could be resolved despite an excess of unbound protein in solution. Fluorescence resonance energy transfer (FRET)-efficiency derived distances for the complexin-SNARE interaction were consistent with the crystal structure of the complexin-SNARE complex. The unstructured N-terminal region of complexin showed broad distributions of FRET efficiencies to the SNARE complex, suggesting that information on conformational variability can be obtained from FRET efficiency distributions. The low-affinity interaction of synaptotagmin with the SNARE complex changed dramatically upon addition of Ca2+ with high FRET efficiency interactions appearing between the C2B domain and linker domains of synaptotagmin and the membrane proximal portion of the SNARE complex. These results demonstrate that single molecule FRET can be used as a "spectroscopic ruler" to simultaneously gain structural and kinetic information about transient multiprotein complexes at the membrane interface.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

[Study on conformational changes of GP IIb/IIIa complex during platelet activation].

OBJECTIVE: To determine whether a conformational change in GPIIb/IIIa occurs during platelet activation. METHODS: Epitopes on GPIIb/IIIa complex were labeled with McAbs to GPIIb (SZ-22) and GPIIIa (SZ-21) conjugated to either a donor fluorescein (FITC-labeled) or an acceptor (TR-labeled) chromophore. The FRET between platelet-bound FITC and TR was measured and calculated by flow cytometry during platelet activation induced by some agonists. RESULTS: In unstimulated platelets, a small but consistent change of FRET (5.5%) was detected, regardless of which antibody served as FRET donor. Platelet activation resulted in an obvious increase in FRET, suggesting a significant change in the separation or orientation of the epitopes within GPIIb/IIIa. This kind of changes could also occur on removing the extracellular calcium, and was independent of receptor occupancy. CONCLUSION: The increase in FRET can reflect the rearrangement of the chromophore-labeled McAbs bound to GPIIb/IIIa during platelet activation, and this kind of structural rearrangement subunits of GPIIb/IIIa complex subunits can induce fibrinogen receptor expression.

Flow Cytometry↗
PubMed1975

Nominal standard dose and tumor standard dose. Tables for radiation therapy planning and analysis.

The method of treatment planning for a predetermined NSD value is described in detail using various example-problems. The Fret tables allow the finding of the total number of fractions needed (NT) for the NSD. This is done through the NSD/d ratio, d standing for the fractional dose in rad. The Fret tables are for 1 to 7 fractions-per-week treatment schedules. The corresponding value of T (elapsed days) are shown for different week days of the therapy initiation with their respective Fret and NSD/d values. The handling of the rest and multi-rest periods is described. A method of finding the NSD value for a treatment which has reached the maximum connective tissue tolerance is described, covering even the most complex treatment plans. Fret-tumor tables for NSD-tumor and their use are described by appropriate example-problems. Ret equivalent therapy planning through direct NSD methods (Fret tables) and through an approximation method (tables provided) is described and the usage demonstrated by example-problems. The usage of parallel opposing and multiple portals is evaluated in ret-dose values (peripheral radiobiologic effect) and certain conclusions drawn to guide the therapist. These show in which situations all portals per session should be used and when alternate portals are more beneficial. The effect of portal weighting is included in this analysis. The application of ELLIS' NSD method for radium therapy is described. If, in the future, any changes in the power factors of the present NSD formula become necessary, the basic handling of the NSD problems described in this manuscript will remain unchanged. The values obtained from these tables can then be adjusted by the appropriate factors.

Cobalt Radioisotopes↗
PubMed2005

Use of fluorescence resonance energy transfer for rapid detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates.

SETTING: Rapid detection of drug resistance in Mycobacterium tuberculosis is important to select effective treatment and prevent transmission of resistant isolates. OBJECTIVE: To evaluate the use of fluorescence resonance energy transfer (FRET) for rapid detection of isoniazid (INH) resistance in M. tuberculosis clinical isolates. DESIGN: One hundred INH-resistant and 50 INH-susceptible isolates of M. tuberculosis were included in the study. The drug susceptibility of all isolates was determined by the standard agar proportion method, and all isolates were then tested by FRET. Three genes associated with INH resistance, katG, inhA and ahpC, were analysed. All isolates were amplified with three pairs of primers. Three pairs of fluorescently labelled DNA probes specific to codon 315 of katG, nucleotide 209 in the regulatory region of inhA and a frequent mutation site in the intergenic region of oxyR-ahpC, were used for mutation detection. RESULTS: The results obtained using FRET were compared with those from the proportion method. The sensitivity and specificity of FRET were respectively 76% and 100%. The frequencies of mutations were 48% in katG, 17% in inhA, 8% in ahpC, 2% in inhA-ahpC and 1% in inhA-katG. CONCLUSION: FRET is a rapid, specific method that can be useful to detect INH resistance in M. tuberculosis clinical isolates.

Drug Resistance, Bacterial↗
PubMed1997

Fluorescence resonance energy transfer in studies of inter-chromophoric distances in biomolecules.

Fluorescence resonance energy transfer (FRET) is a technique widely used in studies of interchromophoric distances in biomolecules such as peptides, proteins and nucleic acids. FRET is especially useful in determination of conformational changes caused by a solvent, presence of denaturing agents, diffusion and other external factors. Precision of interchromophoric distances obtained using the FRET technique is comparable with that of low-resolution X-ray diffraction and NMR data. Comparison of FRET results with the crystal structure for several proteins is reviewed. Moreover, the effect of the orientation factor kappa2 value on FRET results and determinants of kappa2 are discussed.

Protein Conformation↗
PubMed2003

Fluorescence resonance energy transfer between donor-acceptor pair on two oligonucleotides hybridized adjacently to DNA template.

We use fluorescein as the energy donor and rhodamine as the acceptor to measure the efficiency of fluorescence resonance energy transfer (FRET) in a set of hybridized DNA constructs. The two fluorophores are covalently attached via linkers to two separate oligonucleotides with fluorescein at the 3' end of one oligonucleotide and rhodamine at the 5' end or in the middle of another nucleotide. For the FRET analysis both fluorophore-labeled oligonucleotides are hybridized to adjacent sections of the same DNA template to form a three-component duplex with a one base gap between the two labeled oligonucleotides. A similar configuration is implemented for a quantitative real-time polymerase chain reaction (PCR) with LightCycler technology, where a 1-5 base separation between donor and acceptor is recommended to optimize energy transfer efficiencies. Our constructs cover donor-acceptor separations from 2 to 17 base pairs (approximately 10-70 A). The results show that, when the two fluorophores are located at close distances (less than 8 base separation), FRET efficiencies are above 80%, although there may be ground-state interactions between fluorophores when the separation is under about 6 bases. Modeling calculations are used to predict the structure of these three-component constructs. The duplex mostly retains a normal double helical structure, although slight bending may occur near the unpaired base in the DNA template. Stable and reproducible energy transfer is also observed over the distance range investigated here in real-time thermal cycling. The study identifies important parameters that determine FRET response in applications such as real-time PCR.

Base Sequence↗