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At least 55 records · Page 3Linked to original sources
PubMed1996

Cyanine dyes with high absorption cross section as donor chromophores in energy transfer primers.

Energy transfer (ET) fluorescent primers are significantly superior to single dye-labeled primers for DNA sequencing and multiplex genetic analyses (Ju, J., Glazer, A. N., and Mathies, R. A. (1996) Nature Med. 2, 246-249). We describe here ET primers in which a donor chromophore with a large absorption cross section but a low fluorescence quantum yield is exploited to increase the Stokes-shifted fluorescence emission of acceptor dyes. The new ET primers have 3-(epsilon-carboxy-pentyl)-3'ethyl-5,5'-dimethyloxacarbocyanine (CYA; epsilon M488nm 142,000 M-1 cm-1) at the 5' -end as a common energy donor, and fluorescein or rhodamine derivatives (FAM, R6G, TAMRA, and ROX), attached to a modified thymidine 10 bases away within the primer sequence, as acceptors. With 488-nm excitation, the fluorescence emission intensity of these four ET primers is 1.4- to 24-fold stronger than that of the corresponding primers labeled only with the single acceptor dye. When compared with the corresponding ET primers with a fluorescein derivative (FAM; epsilon M488nm 60,000 M-1 cm-1) as donor, the fluorescence emissions of primers with CYA as donor and FAM, R6G, TAMRA, and ROX as acceptors are respectively 0.8-, 1.0-, 1.7-, and 1.7-fold as intense. The low fluorescence quantum yield of the CYA donor resulted in distinct fluorescence signals for the DNA-sequencing fragments with much lower crosstalk between the four detection channels than that seen with ET primers based on a FAM donor. With single-stranded M13mp18 DNA as the template, the CYA ET primers provided DNA sequences on a four-color capilary sequencer with 100% accuracy in the first 500 bases.

Carbocyanines↗
PubMed1998

Detection of programmed cell death using fluorescence energy transfer.

Fluorescence energy transfer (FRET) can be generated when green fluorescent protein (GFP) and blue fluorescent protein (BFP) are covalently linked together by a short peptide. Cleavage of this linkage by protease completely eliminates FRET effect. Caspase-3 (CPP32) is an important cellular protease activated during programmed cell death. An 18 amino acid peptide containing CPP32 recognition sequence, DEVD, was used to link GFP and BFP together. CPP32 activation can be monitored by FRET assay during the apoptosis process.

Amino Acid Sequence↗
PubMed1981

Proximity relationships within the Fc segment of rabbit immunoglobulin G analyzed by resonance energy transfer.

Resonance energy transfer analysis has been carried out with a noncovalent rabbit hybrid of immunoglobulin G (IgG) composed of normal rabbit IgG and rabbit anti-lactose IgG. The hybrid IgG was prepared from proteins in which the single inter-heavy-chain disulfide linkage was specifically reduced and alkylated. Normal rabbit IgG was alkylated with the iodoacetyl derivative of N-(aminoethyl)-5-naphthylamine-1-sulfonic acid while the rabbit anti-lactose IgG was alkylated with either iodoacetamide or the iodoacetyl derivative of p-[[p-(dimethylamino)phenyl]azo]aniline. Fractionation with an anti-lactose-specific immunoadsorbent yielded a population in which each fluorescent donor [N-[(acetylamino)ethyl]-5-naphthylamine-1-sulfonic acid] was adjacent to a nonfluorescent acceptor [N-acetyl-p[[p-dimethylamino)phenyl]. Data on fluorescence quantum yield, excited-state lifetime, time-resolved emission anisotropy, and steady-state fluorescence polarization revealed a distribution of distances between the donor and acceptor. In the native molecule, the hinge regions are known to be covalently liked by a single disulfide bond. In the absence of this linkage, the hinge regions were separated such that for a majority of the molecules in solution (approximately 60%) the separation was 50-60 A. In the context of current knowledge of the IgG molecule, it is evident that the principle forces maintaining the integrity of the native, functional Fc segment are the strong noncovalent interactions of the CH3 domains and the single inter-heavy-chain disulfide bond.

Animals↗
PubMed2005

A new fluorescence resonance energy transfer pair and its application to oligonucleotide labeling and fluorescence resonance energy transfer hybridization studies.

We describe two new fluorescence resonance energy transfer (FRET) compatible labels, their covalent linkage to oligonucleotides, and their use as donor and acceptor, respectively, in FRET hybridization studies. The dyes belong to the cyanine dyes, and water solubility is imparted by a phosphonate which represents a new solubilizing group in DNA labels. They were linked to amino-modified synthetic oligonucleotides via oxysuccinimide (OSI) esters. The studies performed include binding assays, determinations of molecular distances, homogeneous competitive assays, and limits of detection, which are in the order of 5 pmol/L for a 15-mer.

Journal Article↗
PubMed2002

Pathways for energy transfer in the core light-harvesting complexes CP43 and CP47 of photosystem II.

The pigment-protein complexes CP43 and CP47 transfer excitation energy from the peripheral antenna of photosystem II toward the photochemical reaction center. We measured the excitation dynamics of the chlorophylls in isolated CP43 and CP47 complexes at 77 K by time-resolved absorbance-difference and fluorescence spectroscopy. The spectral relaxation appeared to occur with rates of 0.2-0.4 ps and 2-3 ps in both complexes, whereas an additional relaxation of 17 ps was observed only in CP47. Using the 3.8-A crystal structure of the photosystem II core complex from Synechococcus elongatus (A. Zouni, H.-T. Witt, J. Kern, P. Fromme, N. Krauss, W. Saenger, and P. Orth, 2001, Nature, 409:739-743), excitation energy transfer kinetics were calculated and a Monte Carlo simulation of the absorption spectra was performed. In both complexes, the rate of 0.2-0.4 ps can be ascribed to excitation energy transfer within a layer of chlorophylls near the stromal side of the membrane, and the slower 2-3-ps process to excitation energy transfer to the calculated lowest excitonic state. We conclude that excitation energy transfer within CP43 and CP47 is fast and does not contribute significantly to the well-known slow trapping of excitation energy in photosystem II.

Cyanobacteria↗
PubMed1988

Distribution of phospholipids around gramicidin and D-beta-hydroxybutyrate dehydrogenase as measured by resonance energy transfer.

A resonance energy transfer method was developed to study the distribution of phospholipids around integral membrane proteins. The method involved measuring the extent of energy transfer from tryptophan residues of the proteins to different phospholipids labeled with a dansyl moiety in the fatty acid chain. No specific interactions were observed between gramicidin and dansyl-labeled phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid. The results were consistent with a random distribution of each phospholipid in the bilayer in the presence of gramicidin. However, a redistribution of both gramicidin and dansyl-labeled phospholipids was easily observed when a phase separation was induced by adding Ca2+ to vesicles made up of phosphatidylcholine and phosphatidic acid. Polarization measurements showed that in the presence of Ca2+ a rigid phosphatidic acid rich region and a more fluid phosphatidylcholine-rich region were formed. Energy-transfer measurements from gramicidin to either dansylphosphatidylcholine or dansylphosphatidic acid showed gramicidin preferentially partitioned into the phosphatidylcholine-rich regions. Energy-transfer measurements were also carried out with D-beta-hydroxybutyrate dehydrogenase reconstituted in a vesicle composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid. Although the enzyme has a specific requirement for phosphatidylcholine for activity, the extent of energy transfer decreased in the order dansylphosphatidic acid, dansylphosphatidylcholine, dansylphosphatidylethanolamine. Thus, the enzyme reorganized the phospholipids in the vesicle into a nonrandom distribution.

Dansyl Compounds↗
PubMed1994

Does slow energy transfer limit the observed time constant for radical pair formation in photosystem II reaction centers?

We have used spectrally photoselective femtosecond transient absorption spectroscopy on photosystem II reaction centers to show that there are at least two pools of chlorin molecules/states which can transfer excitation energy to P680, the primary electron donor in photosystem II. It has previously been shown that one chlorin pool equilibrates with P680 in 100 fs [Durrant et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11632-11636], and we report here the observation of energy transfer from a second more weakly coupled chlorin pool. The effect of the weakly coupled pool is to increase the apparent time constant for radical pair formation from 21 ps when P680 is selectively excited to 27 ps when the accessory chlorins are excited. We conclude that it is possible to observe both radical pair formation somewhat slowed by an energy transfer step and radical pair formation not limited by this slow energy transfer, depending upon which chromophores are initially excited. These observations provide evidence that when using photoselective excitation of P680, the observed 21 ps time constant for radical pair formation is not limited by a slow energy transfer step.

Energy Transfer↗
PubMed1993

Calculation of photon mass energy-transfer and mass energy-absorption coefficients.

Calculations of mass energy-transfer and mass energy-absorption coefficients for photon energies from 1 keV to 100 MeV have been developed, based on a re-examination of the processes involved after the initial photon interaction. The probabilities for the initial interaction are from the current photon interaction cross-section database at the National Institute of Standards and Technology. The calculations then take into account (1) electron binding effects on the Compton-scattered photon distribution; (2) the complete cascade of fluorescence emission after ionization events in any atomic subshell, including those associated with incoherent scattering and triplet production; and (3) the radiative energy losses of the secondary electrons and positrons slowing down in the medium, including the emission of bremsstrahlung, characteristic X rays from impact ionization, and positron in-flight as well as at-rest annihilation quanta. Consideration of the processes in (3) goes beyond the continuous-slowing-down approximation and includes the effects of energy-loss straggling. Results for the mass energy-absorption coefficient are compared with those from recent tabulations.

Absorption↗
PubMed2002

Spectroscopy of single phycoerythrocyanin monomers: dark state identification and observation of energy transfer heterogeneities.

Phycoerythrocyanin (PEC) is part of the light harvesting system of cyanobacteria. The PEC monomer contains one phycoviolobilin chromophore, which transfers excitation energy onto two phycocyanobilin chromophores. Many spectroscopical methods have been used in the past to study the bulk properties of PEC. These methods average over many molecules. Therefore, differences in the behavior of individual molecules remain hidden. The energy transfer within photosynthetic complexes is however sensitive to changes in the spectroscopic properties of the participating subunits. Knowledge about heterogeneities is therefore important for the description of the energy transfer in photosynthetic systems. Here, the recording of the fluorescence emission of single PEC molecules is used as a tool to obtain such information. Spectrally resolved detection as well as double resonance excitation of single PEC molecules is used to investigate their bleaching behavior. The trans isomer of the phycoviolobilin chromophore is identified as a short-lived dark state of monomeric PEC. Polarization sensitive single molecule detection is used for the direct observation of the energy transfer in individual PEC molecules. The experiments reveal that more than one-half of the PEC molecules exhibit an energy transfer behavior significantly different from the bulk. These heterogeneities persist on a time scale of several seconds. Model calculations lead to the conclusion that they are caused by minor shifts in the spectra of the chromophores.

Biophysical Phenomena↗
PubMed2005

[Determination of vitamin B12 concentration by fluorescence quenching with acridine orange-rhodamine 6G energy transfer system].

An energy transfer technique between acridine orange (AO) and rhodamine 6G (R6G) was studied, and the optimum experimental conditions of energy transfer were defined. It was found that the effective energy transfer could occur between AO and R6G in the dodecylbenzene sodium sulfonate solution with Na2 HPO4-citric acid buffer solution at pH 5.0. The fluorescence intensity of AO-R6G system was diminished by vitamin B12 in an alkalescence medium. Based on the AO-R6G energy transfer system anovel fluorescence quenching method for the determination of vitamin B12 has been developed. Under optimal conditious, the linear range of calibration curves for the determination of vitamin B12 was 0-3.0 x 10(-5) mol x L(-1). The detection limits were 4.8 x 10(-7) mol x L(-1) for Vitamin B12. Among six times of determination, the relative standard deviation was 0.51%-0.64%, and the recovery was 98.40% -103.62%. The method features good recurrence, rapidity of reaction, good stability, and few interfering substances. It can be satisfactorily used in the determination of the injection content of vitamin B12.

Acridine Orange↗
PubMed2006

A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer.

Bioluminescence resonance energy transfer (BRET), which relies on nonradiative energy transfer between luciferase-coupled donors and GFP-coupled acceptors, is emerging as a useful tool for analyzing the quaternary structures of cell-surface molecules. Conventional BRET analyses are generally done at maximal expression levels and single acceptor/donor ratios. We show that under these conditions substantial energy transfer arises from random interactions within the membrane. The dependence of BRET efficiency on acceptor/donor ratio at fixed surface density, or expression level at a defined acceptor/donor ratio, can nevertheless be used to correctly distinguish between well-characterized monomeric and oligomeric proteins, including a very weak dimer. The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein-coupled receptors (GPCRs) proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments.

Binding Sites↗
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↗
PubMed2006

Conversion of intramolecular singlet electron transfer at room temperature into triplet energy transfer at 77 K: photoisomerization in norbornadiene- and carbazole-labeled poly(aryl ether) dendrimers.

A series of Fréchet-type poly(aryl ether) dendrimers (CZ-Gn-NBD, n = 1-3) with carbazole (CZ) chromophores and a norbornadiene (NBD) group attached to the periphery and the core, respectively, were synthesized, and their photophysical and photochemical properties were investigated. Selective excitation of the carbazole units in CZ-Gn-NBD resulted in a singlet electron transfer from CZ to NBD at room temperature, and an intersystem crossing followed a triplet-triplet energy transfer from CZ to NBD in glassy 2-methyltetrahydrofuran at 77 K. Both singlet electron transfer and triplet energy transfer processes lead to the isomerization of the norbornadiene group into the quadricyclane (CZ-Gn-QC). The efficiencies and the rate constants for singlet electron transfer are approximately 88, 80, and 74% and 1.8 x 10(9), 6.1 x 10(8), and 4.0 x 10(8) s(-1) for generations 1-3, respectively. The quantum yields of the intramolecular photosensitized isomerization are measured to be approximately 0.013, 0.012, and 0.011, and the efficiencies of triplet norbornadiene formation via singlet electron transfer are approximately 0.070, 0.065, and 0.059 for generations 1-3, respectively. The light-harvesting ability of CZ-Gn-NBD increases with the generation due to an increase of the number of peripheral chromophores. In glassy 2-methyltetrahydrofuran at 77 K, the triplet-triplet energy transfer proceeds with efficiencies of approximately 0.86, 0.64, and 0.36 and rate constants of 0.96, 0.25, and 0.08 s(-1) for generations 1-3, respectively. The intramolecular singlet electron transfer and triplet energy transfer in CZ-Gn-NBD proceed mainly via a through-space mechanism involving the proximate donor (folding back conformation) and acceptor groups.

Journal Article↗
PubMed1994

Radially restricted linear energy transfer for high-energy protons: a new analytical approach.

Radially restricted linear energy transfer (LET) is a basic physical parameter relevant to radiation biology and radiation protection. In this report a convenient method is presented for the analytical computation of this quantity without the need for complicated simulation. The method uses the energy-restricted LET L delta, as recently redefined in a 1993 ICRU draft document and supplements it by a relatively simple term that represents the energy of fast delta rays lost within distance r from the track core. The method provides a better fit than other models and is valid over the entire range of radial distance from track center to the maximum radial distance traveled by the most energetic secondary electrons. Lr computed by this approach differs only a few percent from the values obtained from explicit Monte Carlo simulations. The concept applies equally to heavy ions and to electrons.

Linear Energy Transfer↗
PubMed1978

Energy transfer and site of energy trapping in photosystem I.

Studies of mutants of Chlamydomonas reinhardi deficient in the chlorophyll . protein complex CP1 show that energy transfer occurs between at least three Photosystem I units. Evidence for the connection of the Photosystem I units is also obtained by studying their optical cross-section in wild-type cells in which a fraction of the Photosystem I centers are inactivated. The site of energy trapping in the Photosystem I unit is shown to be the photochemical center itself and not the surrounding long-wavelength absorbing forms of chlorophyll.

Chlamydomonas↗