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At least 451 records · Page 25Linked to original sources
PubMed1997

Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A.

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor label initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label, R-phycoerythrin and tetramethylrhodamine isothio-cyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.

Animals↗
PubMed1996

Refolding of triosephosphate isomerase in low-water media investigated by fluorescence resonance energy transfer.

The refolding and reassociation of rabbit muscle triosephosphate isomerase (TPI) monomers unfolded with guanidine hydrochloride (GdnHCl) was studied in aqueous media and in reverse micelles (RM) formed with hexadecyltrimethylammonium bromide and n-octane/hexanol. Fluorescence resonance energy transfer (FRET) studies were carried out using TPI labeled at Cys-217 with 5-((2-((iodoacetyl)-amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) and TPI labeled at one of the free amino groups with fluorescein 5'-isothiocyanate (FITC). Efficient FRET between monomers of AEDANS-TPI and FITC-TPI in aqueous media was detected 2-3 min after denaturant dilution and remained constant for hours. The distance between AEDANS and FITC in a labeled, renatured hetero-TPI dimer calculated from FRET results was 48 A, in reasonable agreement with estimates based on the crystal structure of TPI. In RM, recovery of enzyme activity during renaturation correlates with the appearance of a high-intrinsic fluorescence intermediate believed to be a partially folded monomer (Fernández-Velasco et al., 1995). Nevertheless, when AEDANS- and FITC-labeled monomers were mixed in RM, FRET occurred as soon as GdnHCl was diluted (FRET efficiency = 0.36), preceding the changes in TPI intrinsic fluorescence and reactivation. Thereafter, the efficiency of FRET increased during the next hour up to approximately 0.50, where it remained after 24 h, when 80% of the enzyme activity was recovered. The high initial FRET seen in RM could indicate the formation of an inactive dimer within the first minutes after denaturant dilution. The further increase in FRET observed over the next hour could reflect conformational rearrangements of the protein and transition from the inactive to the active dimer.

Animals↗
PubMed2006

Ultrafast fluorescence resonance energy transfer in a micelle.

Ultrafast fluorescence resonance energy transfer (FRET) from coumarin 153 (C153) to rhodamine 6G (R6G) is studied in a neutral PEO(20)-PPO(70)-PEO(20) triblock copolymer (P123) micelle and an anionic micelle (sodium dodecyl sulfate, SDS) using a femtosecond up-conversion setup. Time constants of FRET were determined from the rise time of the acceptor emission. It is shown that a micelle increases efficiency of FRET by holding the donor and the acceptor at a close distance (intramicellar FRET) and also by tuning the donor and acceptor energies. It is demonstrated that in the P123 micelle, intramicellar FRET (i.e., donor and acceptor in same micelle) occurs in 1.2 and 24 ps. In SDS micelle, there are two ultrafast components (0.7 and 13 ps) corresponding to intramicellar FRET. The role of diffusion is found to be minor in the ultrafast components of FRET. We also detected a much longer component (1000 ps) for intramicellar FRET in the larger P123 micelle.

Absorption↗
PubMed2006

Ultrafast fluorescence resonance energy transfer in a reverse micelle: excitation wavelength dependence.

Fluorescence resonance energy transfer (FRET) from coumarin 480 (C480) to fluorescein 548 (F548) in a sodium dioctyl sulfosuccinate (AOT) reverse micelle is studied by picosecond and femtosecond emission spectroscopy. In bulk water, at the low concentration of the donor (C480) and the acceptor (F548), no FRET is observed. However, when the donor (C480) and the acceptor (F548) are confined in a AOT reverse micelle very fast FRET is observed. The time constants of FRET were obtained from the rise time of the emission of the acceptor (F548). In a AOT microemulsion, FRET is found to occur in multiple time scales--3, 200, and 2700 ps. The 3 ps component is assigned to FRET in the water pool of the reverse micelle with a donor-acceptor distance, 16 A. The 200 ps component corresponds to a donor-acceptor distance of 30 A and is ascribed to the negatively charged acceptor inside the water pool and the neutral donor inside the alkyl chains of AOT. The very long 2700 ps component may arise due to FRET from a donor outside the micelle to an acceptor inside the water pool and also from diffusion of the donor from bulk heptane to the reverse micelle. With increase in the excitation wavelength from 375 to 405 nm the relative contribution of the FRET due to C480 in the AOT reverse micelle (the 3 and 200 ps components) increases.

Colloids↗
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↗
PubMed2001

Fluorescence resonance energy transfer studies on the interaction between the lactate transporter MCT1 and CD147 provide information on the topology and stoichiometry of the complex in situ.

The monocarboxylate (lactate) transporters MCT1 and MCT4 require the membrane-spanning glycoprotein CD147 for their correct plasma membrane expression and function. We have successfully expressed CD147 and MCT1 tagged on their C or N termini with either the cyan (CFP) or yellow (YFP) variants of green fluorescent protein. The tagged proteins were correctly targeted to the plasma membrane of COS-7 cells and were functionally active. Measurements of fluorescence resonance energy transfer (FRET) between all combinations of the tagged proteins were made. FRET was observed when either the C or N terminus of MCT1 (intracellular) is tagged with CFP or YFP and co-expressed with CD147 tagged with YFP or CFP on the C terminus (intracellular) but not the N terminus (extracellular). FRET was also observed between two CD147 molecules when both YFP and CFP were on the C terminus but not when both were on the N terminus or one on either end. No FRET was observed between MCT1-YFP and MCT-CFP in any combination. A wide range of controls including photobleaching were employed to confirm that where FRET was observed, it was not an artifact of direct excitation of YFP by the CFP excitation laser. It was also shown that nonspecific overcrowding of proteins did not induce FRET. Because FRET only occurs between two fluorophores if they are less than 100 A apart and in a suitable orientation, our data provide important information on the topology of CD147 and MCT1 within the plasma membrane. The minimum configuration consistent with the data is a dimer of CD147 associating with two MCT1 molecules such that the C terminus of CD147 in the cytosol is close to the C terminus of its partner CD147 and to the C and N termini of an associated MCT1 molecule. FRET may provide a non-invasive technique for measuring changes in these interactions in living cells.

Animals↗
PubMed2005

Role of regulatory F-domain in hepatocyte nuclear factor-4alpha ligand specificity.

The F-domain of rat HNF-4alpha1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4alpha. (i) Fluorescent binding assays indicate that wild-type, full-length HNF-4alpha (amino acids 1-455) has high affinity (Kd=0.06-12 nm) for long chain fatty acyl-CoAs (LCFA-CoA) and low affinity (Kd=58-296 nm) for unesterified long chain fatty acids (LCFAs). LCFA-CoA binding was due to close molecular interaction as shown by fluorescence resonance energy transfer (FRET) from full-length HNF-4alpha tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor), which yielded an intermolecular distance of 33 A, although no FRET to cis-parinaric acid was detected. (ii) Deleting the N-terminal A-D-domains, comprising the AF1 and DNA binding functions, only slightly affected affinities for LCFA-CoAs (Kd=0.9-4 nm) and LCFAs (Kd=93-581 nm). (iii) Further deletion of the F-domain robustly reduced affinities for LCFA-CoA and reversed ligand specificity (i.e. high affinity for LCFAs (Kd=1.5-32 nm) and low affinity for LCFA-CoAs (Kd=54-302 nm)). No FRET from HNF-4alpha-E (amino acids 132-370) tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor) was detected, whereas an intermolecular distance of 28 A was calculated from FRET between HNF-4alpha-E and cis-parinaric acid. (iv) Circular dichroism showed that LCFA-CoA, but not LCFA, altered the secondary structure of HNF-4alpha only when the F-domain was present. (v) cis-Parinaric acid bound to HNF-4alpha with intact F-domain was readily displaceable by S-hexadecyl-CoA, a nonhydrolyzable thioether analogue of LCFA-CoAs. Truncation of the F-domain significantly decreased cis-parinaric acid displacement. Hence, the C-terminal F-domain of HNF-4alpha regulated ligand affinity, ligand specificity, and ligand-induced conformational change of HNF-4alpha. Thus, characteristics of F-domain-truncated mutants may not reflect the properties of full-length HNF-4alpha.

Acyl Coenzyme A↗
PubMed2004

Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo.

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.

Animals↗
PubMed2002

iFRET: an improved fluorescence system for DNA-melting analysis.

Fluorescence resonance energy transfer (FRET) is a powerful tool for detecting spatial relationships between macromolecules, one use of which is the tracking of DNA hybridization status. The process involves measuring changes in fluorescence as FRET donor and acceptor moieties are brought closer together or moved farther apart as a result of DNA hybridization/denaturation. In the present study, we introduce a new version of FRET, which we term induced FRET (iFRET), that is ideally suited for melting curve analysis. The innovation entails using a double-strand, DNA-specific intercalating dye (e.g., SYBR Green I) as the FRET donor, with a conventional FRET acceptor affixed to one of the DNA molecules. The SNP genotyping technique dynamic allele specific hybridization (DASH) was used as a platform to compare iFRET to two alternative fluorescence strategies, namely, the use of the intercalating dye alone and the use of a standard FRET pair (fluorescein as donor, 6-rhodamine as acceptor). The iFRET configuration combines the advantages of intercalating dyes, such as high signal strengths and low cost, with maintaining the specificity and multiplex potential afforded by traditional FRET detection systems. Consequently, iFRET represents a fresh and attractive schema for monitoring interactions between DNA molecules.

Alleles↗
PubMed2003

Fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime imaging microscopy to characterize amyloid-beta plaques.

We describe the implementation of a commercial fluorescence lifetime imaging microscopy (FLIM) instrument used in conjunction with a commercial laser scanning multiphoton microscope. The femtosecond-pulsed near-infrared laser is an ideal excitation source for time-domain fluorescence lifetime measurements. With synchronization from the x-y scanners, fluorescence lifetimes can be acquired on a pixel-by-pixel basis, with high spatial resolution. Multiexponential curve fits for each pixel result in two-dimensional fluorescence resonance energy transfer (FRET) measurements that allow the determination of both proximity of fluorescent FRET pairs, as well as the fraction of FRET pairs close enough for FRET to occur. Experiments are described that characterize this system, as well as commonly used reagents valuable for FRET determinations in biological systems. Constructs of CFP and YFP were generated to demonstrate FRET between this pair of green fluorescent protein (GFP) color variants. The lifetime characteristics of the FRET pair fluorescein and rhodamine, commonly used for immunohistochemistry, were also examined. Finally, these fluorophores were used to demonstrate spatially resolved FRET with senile plaques obtained from transgenic mouse brain. Together these results demonstrate that FLIM allows sensitive measurements of protein-protein interactions on a spatial scale less than 10 nm using commercially available components.

Alzheimer Disease↗
PubMed2004

Polarized fluorescence resonance energy transfer microscopy.

Current methods for fluorescence resonance energy transfer (FRET) microscopy of living cells involve taking a series of images with alternating excitation colors in separate camera exposures. Here we present a new FRET method based on polarization that requires only one camera exposure and thereby offers the possibility for better time resolution of dynamic associations among subcellular components. Polarized FRET (p-FRET) uses a simultaneous combination of excitation wavelengths from two orthogonally polarized sources, along with an emission channel tri-image splitter outfitted with appropriate polarizers, to concurrently excite and collect fluorescence from free donors, free acceptors, and FRET pairs. Based upon the throughput in each emission channel as premeasured on pure samples of each of the three species, decoupling of an unknown sample's three polarized fluorescence images can be performed to calculate the pixel-by-pixel concentrations of donor, acceptor, and FRET pairs. The theory of this approach is presented here, and its feasibility is experimentally confirmed by measurements on mixtures of cyan fluorescent protein (CFP), citrine ((Cit) a yellow fluorescent protein variant), and linked fusion proteins (CFP-L16-Cit, CFP-L7-Cit, CFP-L54-Cit) in living cells. The effects of shot noise, acceptor polarization, and FRET efficiency on the statistical accuracy of p-FRET experimental results are investigated by a noise-simulation program.

Animals↗
PubMedDate not supplied

Fluorescence resonance energy transfer analysis of escherichia coli RNA polymerase and polymerase-DNA complexes.

Fluorescence resonance energy transfer (FRET) is a technique allowing measurements of atomic-scale distances in diluted solutions of macromolecules under native conditions. This feature makes FRET a powerful tool to study complicated biological assemblies. In this report we review the applications of FRET to studies of transcription initiation by Escherichia coli RNA polymerase. The versatility of FRET for studies of a large macromolecular assembly such as RNA polymerase is illustrated by examples of using FRET to address several different aspects of transcription initiation by polymerase. FRET has been used to determine the architecture of polymerase, its complex with single-stranded DNA, and the conformation of promoter fragment bound to polymerase. FRET has been also used as a binding assay to determine the thermodynamics of promoter DNA fragment binding to the polymerase. Functional conformational changes in the specificity subunit of polymerase responsible for the modulation of the promoter binding activity of the enzyme and the mechanistic aspects of the transition from the initiation to the elongation complex were also investigated.

DNA↗
PubMed2002

Use of phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow cytometry: advantages and limitations.

BACKGROUND: This study validates the use of phycoerythrin (PE) and allophycocyanin (APC) for fluorescence energy transfer (FRET) analyzed by flow cytometry. METHODS: FRET was detected when a pair of antibody conjugates directed against two noncompetitive epitopes on the same CD8alpha chain was used. FRET was also detected between antibody conjugate pairs specific for the two chains of the heterodimeric alpha (4)beta(1) integrin. Similarly, the association of T-cell receptor (TCR) with a soluble antigen ligand was detected by FRET when anti-TCR antibody and MHC class I/peptide complexes (< >) were used. RESULTS: FRET efficiency was always less than 10%, probably because of steric effects associated with the size and structure of PE and APC. Some suggestions are given to take into account this and other effects (e.g., donor and acceptor concentrations) for a better interpretation of FRET results obtained with this pair of fluorochromes. CONCLUSIONS: We conclude that FRET assays can be carried out easily with commercially available antibodies and flow cytometers to study arrays of multimolecular complexes.

CD8 Antigens↗
PubMed2005

Detecting protein-protein interaction in live yeast by flow cytometry.

BACKGROUND: The yeast Saccharomyces cerevisiae is the most commonly used organism for studying protein- protein interactions. In this report we demonstrate the use of flow cytometry in observing fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent fusion proteins (CFP and YFP, respectively) as a marker for protein interaction in live yeast cells. Probability binning is also employed to provide a statistical confirmation of our observations. METHODS: We coexpressed CFP and YFP fusions containing the N-terminal transmembrane domain (NTM) of Tom70p in yeast and analyzed FRET in live cells with a multilaser flow cytometer. The Tom70p NTM was previously shown to be sufficient for mitochondrial localization and protein-protein interaction (Millar and Shore, 1994, J Biol Chem 269:12229-12232). RESULTS: FRET was observed only in cells that expressed CFP and YFP fusions that each contained the wild-type NTM. The introduction of mutations previously shown to disrupt NTM interaction eliminated FRET. Probability binning confirmed that differences between the FRET channels of experimental and control samples were statistically and physiologically significant. CONCLUSION: Flow cytometric analysis of FRET in yeast is a powerful technique for studying protein-protein interactions. The use of flow cytometry allows FRET data to be gathered from a large number of individual cells, thus providing important advantages unavailable to other techniques. Its application to yeast presents a new method to a popular system widely used in proteomic studies.

Bacterial Proteins↗
PubMed2000

Fluorescence energy transfer analysis of DNA structures containing several bulges and their interaction with CAP.

DNA molecules with three bulges separated by double-stranded helical sections of B-DNA were constructed to be used as substrates for DNA-protein binding assays. Fluorescence resonance energy transfer (FRET) between dye molecules attached to the 5'-ends of the DNA molecules is used to monitor the protein binding. The A5 bulge, which consists of five unpaired adenine nucleotides, alters the direction of the helical axis by approximately 80 to 90 at every bulge site. Computer molecular modeling facilitated a pre-selection of suitable helix lengths that bring the labeled ends of the three-bulge DNA molecules (60 to 70 base-pairs long) into close proximity. The FRET experiments verified that the labeled ends of the helices of these long molecules were indeed close. A series of FRET experiments was carried out with two A5 and two A7 bulge molecules. The relative positions of the bulges were varied along the central helical DNA sequence (between the bulges) in order to determine the relative angular juxtapositions of the outlying helical arms flanking the central helical region. The global structural features of the DNA molecules are manifested in the FRET data. The FRET experiments, especially those of the two-bulge series, could be interpreted remarkably well with molecular models based on the NMR structure of the A5 bulge. These models assume that the DNA molecules do not undergo large torsional conformational fluctuations at the bulge sites. The magnitude of the FRET efficiency attests to a relatively rigid structure for many of the long 5'-end-labeled molecules. The changes in the FRET efficiency of three-bulge structures containing the specific binding sequence of the catabolite activator protein (CAP) demonstrated significant deformation of the DNA upon binding of CAP. No direct interaction of CAP with the dyes was observed.

Adenine↗
PubMedDate not supplied

Fluorescence resonance energy transfer spectroscopy is a reliable "ruler" for measuring structural changes in proteins. Dispelling the problem of the unknown orientation factor.

Fluorescence resonance energy transfer (FRET) spectroscopy has been widely used to "measure" dimensions either within or between molecules over distances of 10-100A, a range that is well suited to probing protein structure. The resolution of FRET spectroscopy is substantially lower than X-ray diffraction (XRD) but the technique makes up for this deficit by being particularly good at measuring structural changes in proteins. However, absolute distances measured by FRET spectroscopy remain problematical because of what appears to be an unjustified assumption, namely that calculations of FRET distances assume that the probes are able to undergo free, isotropic motion. This uncertainty may be ascribed to an unknown value for the orientation factor, but other factors may also be important. Common sense suggests that a large (300-500 Da) hydrophobic FRET probe covalently bound to an amino acid side chain of a protein can not undergo true rotational freedom. If this is so, the calculated distances would at best be flawed and at worst be meaningless. In this paper we argue that the orientation parameter is no longer an important issue in the determination of distances determined by FRET using peptides and proteins. Furthermore, we suggest that FRET may be a good form of spectroscopy for testing models of F-actin.

Actins↗
PubMed2001

Application of fluorescence resonance energy transfer to the GroEL-GroES chaperonin reaction.

Fluorescence resonance energy transfer (FRET) is a sensitive and flexible method for studying protein-protein interactions. Here it is applied to the GroEL-GroES chaperonin system to examine the ATP-driven dynamics that underlie protein folding by this chaperone. Relying on the known structures of GroEL and GroES, sites for attachment of fluorescent probes are designed into the sequence of both proteins. Because these sites are brought close in space when GroEL and GroES form a complex, excitation energy can pass from a donor to an acceptor chromophore by FRET. While in ideal circumstances FRET can be used to measure distances, significant population heterogeneity in the donor-to-acceptor distances in the GroEL-GroES complex makes distance determination difficult. This is due to incomplete labeling of these large, oligomeric proteins and to their rotational symmetry. It is shown, however, that FRET can still be used to follow protein-protein interaction dynamics even in a case such as this, where distance measurements are either not practical or not meaningful. In this way, the FRET signal is used as a simple proximity sensor to score the interaction between GroEL and GroES. Similarly, FRET can also be used to follow interactions between GroEL and a fluorescently labeled substrate polypeptide. Thus, while knowledge of molecular structure aids enormously in the design of FRET experiments, structural information is not necessarily required if the aim is to measure the thermodynamics or kinetics of a protein interaction event by following changes in the binding proximity of two components.

Chaperonin 10↗
PubMed2006

Two-photon excited fluorescence energy transfer: a study based on oligonucleotide rulers.

The use of two-photon excitation of fluorescence for detection of fluorescence resonance energy transfer (FRET) was studied for a selected fluorescent donor-acceptor pair. A method based on labeled DNA was developed for controlling the distance between the donor and the acceptor molecules. The method consists of hybridization of fluorescent oligonucleotides to a complementary single-stranded target DNA. As the efficiency of FRET is strongly distance dependent, energy transfer does not occur unless the fluorescent oligonucleotides and the target DNA are hybridized. A high degree of DNA hybridization and an excellent FRET efficiency were verified with one-photon excited fluorescence studies. Excitation spectra of fluorophores are usually wider in case of two-photon excitation than in the case of one-photon excitation. This makes the selective excitation of donor difficult and might cause errors in detection of FRET with two-photon excited fluorescence. Different techniques to analyze the FRET efficiency from two-photon excited fluorescence data are discussed. The quenching of the donor fluorescence intensity turned to be the most consistent way to detect the FRET efficiency. The two-photon excited FRET is shown to give a good response to the distance between the donor and the acceptor molecules.

DNA, Single-Stranded↗