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At least 19 recordsLinked to original sources
PubMed1993

Kinetics of structural changes of reconstituted skeletal muscle thin filaments observed by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer was measured between probes attached to Troponin-I and actin in the reconstituted skeletal muscle thin filament. Cys-133 on TnI was labeled with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, and Lys-61 on actin was labeled with fluorescein 5-isothiocyanate. A large difference in the efficiency of resonance energy transfer was observed between the presence (0.55) and absence (0.76) of Ca2+, which corresponds to the change in the distance of 0.53 nm and is in good accordance with a previous report (Miki, M. (1990) Eur. J. Biochem. 187, 155-162). The fluorescence titration curve of this reconstituted thin filament was measured versus free Ca2+ concentration under physiological conditions in the presence of 8 mM MgCl2. The transfer efficiency changed sharply between pCa 6.9 and 5.75, with a midpoint at pCa 6.25, suggesting that a conformational change of thin filaments occurs with a highly cooperative mode. The time course of this conformational change was followed by measuring the fluorescence intensity change (energy transfer efficiency change) of the labeled reconstituted thin filament after rapid decrease or increase of free Ca2+ concentration with a stopped-flow fluorometer. When the free Ca2+ concentration was increased in the presence of 5 mM MgCl2, the fluorescence intensity increased (the transfer efficiency decreased) with a rate constant of 530 +/- 170 s-1 at 20 degrees C. On the other hand, when the free Ca2+ concentration was decreased, the fluorescence intensity decreased with a rate constant of 43 +/- 5 s-1. The results indicate that the conformational change observed by fluorescence resonance energy transfer corresponds well to the regulation mechanism by troponin-tropomyosin on the reconstituted skeletal muscle thin filament.

Actins↗
PubMed2001

RNA conformation and folding studied with fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) results from nonradiative coupling of two fluorophores and reports on distances in the range 10-100 A. It is therefore a suitable probe to determine distances in RNA molecules and define their global structure, to follow kinetics of RNA conformational changes during folding in real time, to monitor ion binding, or to analyze conformational equilibria and assess the thermodynamic stability of tertiary structure conformers. Along with the basic principles of steady-state and time-resolved fluorescence resonance energy transfer measurements, approaches to investigate RNA conformational transitions and folding are described and illustrated with selected examples. The versatility of FRET-based techniques has recently been demonstrated by implementations of FRET in high-throughput screening of potential drugs as well as studies of energy transfer that monitor RNA conformational changes on the single-molecule level.

Diagnostic Imaging↗
PubMed2004

Kinetics of the structural transition of muscle thin filaments observed by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer showed that troponin-I changes the position on an actin filament corresponding to three states (relaxed, closed, and open) of the thin filament (Hai et al. (2002) J. Biochem. 131, 407-418). In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to position 1, 133, or 181 of troponin-I and Cys-374 of actin on reconstituted thin filaments was measured to follow the transition between three states of the thin filament. When the free Ca(2+) concentration was increased, the transition from relaxed to closed states occurred with a rate constant of approximately 500 s(-1). For the reverse transition, the rate constant was approximately 60 s(-1). When myosin subfragment-1 was dissociated from thin filaments in the presence of Ca(2+) by rapid mixing with ATP, the transition from open to closed states occurred with a single rate constant of approximately 300 s(-1). Light-scattering measurements showed that the ATP-induced myosin subfragment-1 dissociation occurred with a rate constant of approximately 900 s(-1). In the absence of Ca(2+), the transition from open to relaxed states occurred with two rate constants of approximately 400 and approximately 80 s(-1). These transition rates are fast enough to allow the spatial rearrangement of thin filaments to be involved in the regulation mechanism of muscle contraction.

Actin Cytoskeleton↗
PubMed2006

Distance determination in protein-DNA complexes using fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) provides distance information between a donor and an acceptor dye in the range of 10-100 A. Knowledge of the exact positions of some dyes (e.g., fluorescein, rhodamine, or Cy3) with respect to nucleic acids and DNA design enables us to translate these data into precise structural information using molecular modeling. Here we describe this in vitro approach from the design and synthesis of the DNA FRET samples to the fluorescence spectroscopy methods and analysis. Advances in the preparation of dye-labeled nucleic acid molecules and modern techniques like the measurement of FRET in vivo lead to an increased importance of FRET studies in structural and molecular biology.

DNA↗
PubMed2003

Characterization of DNA/lipid complexes by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) is a potential method for the characterization of DNA-cationic lipid complexes (lipoplexes). In this work, we used FRET models assuming a multilamellar lipoplex arrangement. The application of these models allows the determination of the distance between the fluorescent intercalator on the DNA and a membrane dye on the lipid, and/or the evaluation of encapsulation efficiencies of this liposomal vehicle. The experiments were carried out in 1,2-dioleoyl-3-trimethylammonium-propane/pUC19 complexes with different charge ratios. We used 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and 2-(4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC) as membrane dyes, and ethidium bromide (EtBr) and BOBO-1 as DNA intercalators. In cationic complexes (charge ratios (+/-) >or= 2), we verified that BOBO-1 remains bound to DNA, and FRET occurs to the membrane dye. This was also confirmed by anisotropy and lifetime measurements. In complexes with all DNA bound to the lipid (charge ratio (+/-) = 4), we determined 27 A as the distance between the donor and acceptor planes (half the repeat distance for a multilamellar arrangement). In complexes with DNA unbound to the lipids (charge ratio (+/-) = 0.5 and 2), we calculated the encapsulation efficiencies. The presented FRET methodology is, to our knowledge, the first procedure allowing quantification of lipid-DNA contact.

Binding Sites↗
PubMed2001

Clustering of peptide-loaded MHC class I molecules for endoplasmic reticulum export imaged by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer between cyan fluorescent protein- and yellow fluorescent protein-tagged MHC class I molecules reports on their spatial organization during assembly and export from the endoplasmic reticulum (ER). A fraction of MHC class I molecules is clustered in the ER at steady state. Contrary to expectations from biochemical models, this fraction is not bound to the TAP. Instead, it appears that MHC class I molecules cluster after peptide loading. This clustering points toward a novel step involved in the selective export of peptide-loaded MHC class I molecules from the ER. Consistent with this model, we detected clusters of wild-type HLA-A2 molecules and of mutant A2-T134K molecules that cannot bind TAP, but HLA-A2 did not detectably cluster with A2-T134K at steady state. Lactacystin treatment disrupted the HLA-A2 clusters, but had no effect on the A2-T134K clusters. However, when cells were fed peptides with high affinity for HLA-A2, mixed clusters containing both HLA-A2 and A2-T134K were detected.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Molecular topography imaging by intermembrane fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) between lipid-linked donor and acceptor molecules in two apposing lipid bilayer membranes is used to resolve topographical features at an intermembrane junction. Efficient energy transfer occurs when the membranes are apposed closely, which creates an image, or footprint, that maps the contact zone and reveals nanometer-scale topographical structures. We experimentally characterize intermembrane FRET by using a supported membrane junction consisting of a glass-supported lipid membrane, onto which a second membrane is deposited by rupture of a giant vesicle. A series of membrane junctions containing different glycolipids (phosphatidylinositol and ganglioside G(M1)), protein (cholera toxin), and lipid-linked polyethylene glycol are studied. The carbohydrate and protein components influence the intermembrane separation. Differential FRET efficiency is clearly distinguishable for each case. Quantitative analysis of the FRET efficiency yields measurements of intermembrane-separation distances that agree precisely with structural data on G(M1) and cholera toxin. The lateral arrangement of molecular species on the membrane surface thus can be discerned by their influence on membrane spacing without the need for direct labeling of the molecule of interest. In the case of polyethylene glycol lipid-containing membrane junctions, imaging by intermembrane FRET reveals spontaneously forming patterns that are not visible in conventional fluorescence images.

Cholera Toxin↗
PubMed2004

Measurement of intermolecular distances for the natural agonist Peptide docked at the cholecystokinin receptor expressed in situ using fluorescence resonance energy transfer.

Fluorescence resonance energy transfer is a powerful biophysical technique used to analyze the structure of membrane proteins. Here, we used this tool to determine the distances between a distinct position within a docked agonist and a series of distinct sites within the intramembranous confluence of helices and extracellular loops of the cholecystokinin (CCK) receptor. Pseudo-wild-type CCK receptor constructs having single reactive cysteine residues inserted into each of these sites were developed. The experimental strategy included the use of the full agonist, Alexa488-CCK, bound to these receptors as donor, with Alexa568 covalently bound to the specific sites within the CCK receptor as acceptor. Site-labeling was achieved by derivatization of intact cells with a novel fluorescent methanethiosulfonate reagent. A high degree of spectral overlap was observed between receptor-bound donor and receptor-derivatized acceptors, with no transfer observed for a series of controls representing saturation of the receptor binding site with nonfluorescent ligand and use of a null-reactive CCK receptor construct. The measured distances between the fluorophore within the docked agonist and the sites within the first (residue 102) and third (residue 341) extracellular loops of the receptor were shorter than those directed to the second loop (residue 204) or to intramembranous helix two (residue 94). These distances were accommodated well within a refined molecular model of the CCK-occupied receptor that is fully consistent with all existing structure-activity and photoaffinity-labeling studies. This approach provides the initial insights into the conformation of extracellular loop regions of this receptor and establishes clear differences from analogous loops in the rhodopsin crystal structure.

Amino Acid Sequence↗
PubMed2000

Interaction of myosin LYS-553 with the C-terminus and DNase I-binding loop of actin examined by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) experiments were carried out in the absence of nucleotide (rigor) or in the presence of MgADP between fluorescent donor probes (IAEDANS (5((((2-iodoacetyl)amino)ethyl)amino)-naphthalene-1-sulfonic acid) at Cys-374 or DANSYL (5-dimethylamino naphthalene-1-(N-(5-aminopentyl))sulfonamide) at Gln-41 of actin and acceptor molecules (FHS (6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester) at Lys-553 of skeletal muscle myosin subfragment 1. The critical Förster distance (R(0)) was determined to be 44 and 38 A for the IAEDANS-FHS and DANSYL-FHS donor-acceptor pairs, respectively. The efficiency of energy transfer between the acceptor molecules at Lys-553 of myosin and donor probes at Cys-374 or Gln-41 of actin was calculated to be 0.78 +/- 0.01 or 0.94 +/- 0.01, respectively, corresponding to distances of 35.6 +/- 0.4 A and 24.0 +/- 1.6 A, respectively. MgADP had no significant effect on the distances observed in rigor. Thus, rearrangements in the acto-myosin interface are likely to occur elsewhere than in the lower 50-kDa subdomain of myosin as its affinity for actin is weakened by MgADP binding.

Actins↗
PubMed1996

Determination of DNA helical handedness by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) has been used to determine the helical handedness, twist and rise of different DNA conformations. The approach is based on the construction of a set of molecules consisting of two fused helical segments, one of which is in a known reference helical form. The duplexes are covalently labeled at one end with a donor and at the other with an acceptor. By systematically shifting the position of the junction while maintaining constant the total length in base-pairs, the variation in the efficiency of energy transfer can be shown to depend primarily and sensitively on the differences in helical twist and rise of the two constituent segments. If the latter have the same helical sense, one predicts a FRET signal that is a monotonic function of the junctional position. In contrast, a periodic function arises when two segments are of opposite handedness. The formalism includes explicit consideration of dye orientation (the dipole-dipole orientation factor kappa) and an implementation valid for single helix molecules, and introduces new functions of measured fluorescence signals for establishing the FRET efficiency. The method has been applied to a family of oligonucleotides forming hairpin duplexes containing an antiparallel-stranded (aps) d(m5C.G)m segment labeled at the 5' end with fluorescein (donor) and a second parallel- stranded d(A.T)N-m segment (psAt-DNA) labeled at the hairpin loop with the sulfoindocyanine dye Cy3. The segment lengths were in the range 4 to 12, but the total length N was maintained constant at 16. The d(m5C.G) sequence was chosen due to its capacity for adopting a B or a Z conformation at low and high concentrations of salt, respectively. The parallel-stranded d(A.T) sequence served as the second segment in order to determine the helical rise and twist of psAT-DNA, presumed to be right-handed from molecular modeling and a prior study of topologically constrained DNA. A Z-DNA/ps-DNA junction was created between the two segments by inducing a B-Z transition in d(m5C.G)m with MgCl2. The range of required salt concentration was established by circular dichroism measurements. FRET efficiency values of 0.38 to 0.41 were obtained for the oligonucleotides with the d(m5C.G) segment in the B conformation. In contrast, upon induction of the B-Z transition the FRET efficiency was a decreasing function of the d(m5C.G) content (0.38 to 0.28 for m = 6 to 12). Helical parameters were estimated from functional fits of the data, and were consistent with the known properties of B- and Z-DNAs and with the conclusion that psAT-DNA has a helical rise and twist close to that of B-DNA. The approach outlined here is not restricted to DNA but can be applied to other helical structures, e.g. RNA, proteins, and protein-nucleic acid complexes.

DNA↗
PubMed1994

Kinking of DNA and RNA helices by bulged nucleotides observed by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) has been used to demonstrate the bending of DNA and RNA helices for three series of double-stranded molecules containing bulge loops of unopposed adenosine nucleotides (An, n = 0-9). Fluorescein and rhodamine were covalently attached to the 5' termini of the two component strands. Three different methods were applied to measure the FRET efficiencies. The extent of energy transfer within each series increases as the number of bulged nucleotides varies from 1 to 7, indicating a shortening of the end-to-end distance. This is consistent with a bending of DNA and RNA helices that is greater for larger bulges. The FRET efficiency for DNA molecules with A9 bulges is lower than the efficiency for the corresponding A7 bulged molecules, although the A9 molecules exhibit increased electrophoretic retardation. Ranges of bending angles can be estimated from the FRET results.

Adenine Nucleotides↗
PubMed2001

Single-molecule fluorescence resonance energy transfer.

Fluorescent resonance energy transfer (FRET) is a powerful technique for studying conformational distribution and dynamics of biological molecules. Some conformational changes are difficult to synchronize or too rare to detect using ensemble FRET. FRET, detected at the single-molecule level, opens up new opportunities to probe the detailed kinetics of structural changes without the need for synchronization. Here, we discuss practical considerations for its implementation including experimental apparatus, fluorescent probe selection, surface immobilization, single-molecule FRET analysis schemes, and interpretation.

Energy Transfer↗
PubMed2001

Structural study of DNA duplexes containing the (6-4) photoproduct by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) experiments have been performed to elucidate the structural features of oligonucleotide duplexes containing the pyrimidine(6-4)pyrimidone photoproduct, which is one of the major DNA lesions formed at dipyrimidine sites by UV light. Synthetic 32mer duplexes with and without the (6-4) photoproduct were prepared and fluorescein and tetramethylrhodamine were attached, as a donor and an acceptor, respectively, to the aminohexyl linker at the C5 position of thymine in each strand. Steady-state and time-resolved analyses revealed that both the FRET efficiency and the fluorescence lifetime of the duplex containing the (6-4) photoproduct were almost identical to those of the undamaged duplex, while marked differences were observed for a cisplatin-modified duplex, as a model of kinked DNA. Lifetime measurements of a series of duplexes containing the (6-4) photoproduct, in which the fluorescein position was changed systematically, revealed a small unwinding at the damage site, but did not suggest a kinked structure. These results indicate that formation of the (6-4) photoproduct induces only a small change in the DNA structure, in contrast to the large kink at the (6-4) photoproduct site reported in an NMR study.

Cisplatin↗
PubMed2001

Coexisting conformations of fibronectin in cell culture imaged using fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) between fluorophores attached to single proteins provides a tool to study the conformation of proteins in solution and in cell culture. As a protein unfolds, nanometer-scale increases in distance between donor and acceptor fluorophores cause decreases in FRET. Here we demonstrate the application of FRET to imaging coexisting conformations of fibronectin (Fn) in cell culture. Fn is a flexible 440-kDa extracellular matrix protein, with functional sites that are regulated by unfolding events. Fn was labeled with multiple donor and acceptor fluorophores such that intramolecular FRET could be used to distinguish a range of Fn conformations. The sensitivity of FRET to unfolding was tested by progressively denaturing labeled Fn using guanidium chloride. To investigate Fn conformation changes during cell binding and matrix assembly, we added labeled Fn to the culture medium of NIH 3T3 fibroblasts. Coexisting conformations of Fn were visualized using fluorescence microscopy, and spectra from specific features were measured with an attached spectrometer. Using FRET as an indicator of Fn conformation, Fn diffusely bound to cells was in a compact state, whereas Fn in matrix fibrils was highly extended. Matrix fibrils exhibited a range of FRET that suggested some degree of unfolding of Fn's globular modules. Fn in cell-associated clusters that preceded fibril formation appeared more extended than diffuse cell-bound Fn but less extended than fibrillar Fn, suggesting that Fn undergoes extension after cell binding and before polymerization. FRET thus provides an approach to gain insight into the integrin-mediated pathway of Fn fibrillogenesis.

3T3 Cells↗
PubMed2006

Detecting RNA/DNA hybridization using double-labeled donor probes with enhanced fluorescence resonance energy transfer signals.

Fluorescence resonance energy transfer (FRET) occurs when two fluorophores are in close proximity, and the emission energy of a donor fluorophore is transferred to excite an acceptor fluorophore. Using such fluorescently labeled oligonucleotides as FRET probes, makes possible specific detection of RNA molecules even if similar sequences are present in the environment. A higher ratio of signal to background fluorescence is required for more sensitive probe detection. We found that double-labeled donor probes labeled with BODIPY dye resulted in a remarkable increase in fluorescence intensity compared to single-labeled donor probes used in conventional FRET. Application of this double-labeled donor system can improve a variety of FRET techniques.

DNA↗
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

Detection of DNA hybridization using induced fluorescence resonance energy transfer.

Induced fluorescence resonance energy transfer (iFRET) is a variation of resonance energy transfer that is particularly well-suited for the detection of DNA hybridization. The underlying mechanism involves monitoring changes in fluorescence that are the result of an energy transfer reaction between a specific pair of donor and acceptor moieties. In iFRET, the donor is a dye that only fluoresces while interacting with double-stranded DNA and the acceptor is dye that is covalently linked to an oligonucleotide probe. Hybridization of the probe to its complement induces excitement of the donor dye and subsequent energy transfer to the acceptor dye. The energy transfer reaction (and concomitant hybridization status) can easily be followed by monitoring the fluorescence output of the acceptor dye. Because the interaction of the donor dye is reversible and dependent on the presence of double-stranded DNA, iFRET is extremely useful and herein demonstrated in the generation of DNA melting curves.

DNA↗