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1.
The efficiency of fluorescence resonance energy transfer (FRET) between two chromophores positioned at opposite ends of DNA base pair domains has been investigated. The base pair domain serves as a helical scaffold which defines both the distance between chromophores and the dihedral angle between their electronic transition dipole moments, each incremental base pair increasing the distance and stepping the dihedral angle. Fluorescence quantum yields and lifetimes have been determined for both the donor and acceptor chromophores. The experimental data are found to be in excellent accord with an oriented dipole model, rather than with the averaged dipole model conventionally assumed for FRET. 相似文献
2.
Maus M De R Lor M Weil T Mitra S Wiesler UM Herrmann A Hofkens J Vosch T Müllen K De Schryver FC 《Journal of the American Chemical Society》2001,123(31):7668-7676
Intramolecular F?rster-type excitation energy transfer (FRET) processes in a series of first-generation polyphenylene dendrimers substituted with spatially well-separated peryleneimide chromophores and a terryleneimide energy-trapping chromophore at the rim were investigated by steady-state and time-resolved fluorescence spectroscopy. Energy-hopping processes among the peryleneimide chromophores are revealed by anisotropy decay times of 50--80 ps consistent with a FRET rate constant of k(hopp) = 4.6 ns(-1). If a terryleneimide chromophore is present at the rim of the dendrimer together with three peryleneimide chromophores, more than 95% of the energy harvested by the peryleneimide chromophores is transferred and trapped in the terryleneimide. The two decay times (tau(1) = 52 ps and tau(2) = 175 ps) found for the peryleneimide emission band are recovered as rise times at the terryleneimide emission band proving that the energy trapping of peryleneimide excitation energy by the terryleneimide acceptor occurs via two different, efficient pathways. Molecular- modeling-based structures tentatively indicate that the rotation of the terryleneimide acceptor group can lead to a much smaller distance to a single donor chromophore, which could explain the occurrence of two energy-trapping rate constants. All energy-transfer processes are quantitatively describable with F?rster energy transfer theory, and the influence of the dipole orientation factor in the F?rster equation is discussed. 相似文献
3.
The power of FRET to study molecular complexes is expanded by the use of two or more donor/acceptor pairs. A general theoretical framework for distance measurements in three-chromophore systems is presented. Three energy transfer schemes applicable to many diverse situations are considered: (I) two-step FRET relay with FRET between the first and second chromophores and between the second and third, (II) FRET from a single donor to two different acceptors, and (III) two-step FRET relay with FRET also between the first and third chromophores. Equations for the efficiencies involving multiple energy transfer steps are derived for both donor quenching and sensitized emission measurements. The theory is supported by experimental data on model systems of known structure using steady-state donor quenching, lifetime quenching, and sensitized emission. The distances measured in the three-chromophore systems agree with those in two-chromophore systems and molecular models. Finally, labeling requirements for diagnosis of the energy transfer scheme and subsequent distance measurements are discussed. 相似文献
4.
Ultra time-resoived huorescence spectra were used to study the energy transter processes and mechanism of complex PEC/PC/APC at 77 K, which was reconstructed with phycobiliproteins (PEC, PC and APC) of Anabaena variabilis, and has intact light-harvesting system and single terminal emitter. The energy transfer relationships between different chromophores especially between rod and core were also discussed based on fluorescence decay kinetic under different detected wavelengths. As a result, we got the possible energy transfer pathways and transfer time constants to be 29 ps between two PEC trimers, 12 ps between PEC and C-PC, 51 ps between rod and core. 相似文献
5.
C-Phycocyanin (PC) trimers associated with linker polypeptides were isolated from the phycobilisome (PBS) rods of Synechococcus sp. PCC 7002. LXY refers to a linker polypeptide (L) having an apparent mass of Y kDa, located at position X in the phycobilisome where X can be R (rod), C (core) or RC (rod-core junction). Measurements of the absorption, fluorescence and excitation anisotropy of PC trimer, PC.LR32.3 and PC.LRC28.5 complexes document the spectroscopic modulation of each linker polypeptide on the PC chromophores. The difference spectra between the PC trimer and the PC-linker complexes show that although the effect induced by the linker polypeptides is qualitatively similar in behavior, the extent of the modulation is greater in PC.LRC28.5. Measurements taken at 77 K show that a red-wavelength component of the PC trimer absorption-fluorescence spectra is the target of the linker's influence and that this component is altered to a greater extent by LRC28.5. In addition the 77 K absorbance of the PC trimer resolves band features that are consistent with an excitonic coupling interaction between neighboring alpha 84 and beta 84 chromophores. These band features are also evident in the absorbance of PC.LR32.3 but are absent in PC.LRC28.5 indicating that LRC28.5 may be perturbing the coupling interaction established in the PC trimer alpha 84-beta 84 chromophore pairs. Structurally, the linker polypeptide should disrupt the C3 symmetry in the central cavity of the associated phycobiliprotein and this asymmetric interaction should serve to guide the transfer of excitation energy along PBS rods toward the core elements. 相似文献
6.
Rindermann JJ Akhtman Y Richardson J Brown T Lagoudakis PG 《Journal of the American Chemical Society》2011,133(2):279-285
Intramolecular distances in proteins and other biomolecules can be studied in living cells by means of fluorescence resonance energy transfer (FRET) in steady-state or pulsed-excitation experiments. The major uncertainty originates from the unknown orientation between the optical dipole moments of the fluorescent markers, especially when the molecule undergoes thermal fluctuations in physiological conditions. We introduce a statistical method based on the von Mises-Fisher distribution for the interpretation of fluorescence decay dynamics in donor-acceptor FRET pairs that allows us to retrieve both the orientation and the extent of directional fluctuations of the involved dipole moments. We verify the method by applying it to donor-acceptor pairs controllably attached to DNA helices and find that common assumptions such as complete rotational freedom or fully hindered rotation of the dipoles fail a physical interpretation of the fluorescence decay dynamics. This methodology is applicable in single-molecule and ensemble measurements of FRET to derive more accurate distance estimates from optical experiments, without the need for more complex and expensive NMR studies. 相似文献
7.
Hannestad JK Sandin P Albinsson B 《Journal of the American Chemical Society》2008,130(47):15889-15895
DNA is a promising material for use in nanotechnology; the persistence length of double stranded DNA gives it a rigid structure in the several-nanometer regime, and its four letter alphabet enables addressability. We present the construction of a self-assembled DNA-based photonic wire capable of transporting excitation energy over a distance of more than 20 nm. The wire utilizes DNA as a scaffold for a chromophore with overlapping absorption and emission bands enabling fluorescence resonance energy transfer (FRET) between pairs of chromophores leading to sequential transfer of the excitation energy along the wire. This allows for the creation of a self-assembled photonic wire using straightforward construction and, in addition, allows for a large span in wire lengths without changing the basic components. The intercalating chromophore, YO, is chosen for its homotransfer capability enabling effective diffusive energy migration along the wire without loss in energy. In contrast to heterotransfer, i.e., multistep cascade FRET, where each step renders a photon with less energy than in the previous step, homotransfer preserves the energy in each step. By using injector and detector chromophores at opposite ends of the wire, directionality of the wire is achieved. The efficiency of the wire constructs is examined by steady-state and time-resolved fluorescence measurements and the energy transfer process is simulated using a Markov chain model. We show that it is possible to create two component DNA-based photonic wires capable of long-range energy transfer using a straightforward self-assembly approach. 相似文献
8.
Cheng‐Lan Lin Dr. Chih‐Hsien Chen Tsong‐Shin Lim Prof. Wunshain Fann Prof. Tien‐Yau Luh Prof. 《化学:亚洲杂志》2008,3(3):578-584
A convenient protocol to fabricate an organic–inorganic hybrid system with covalently bound light‐harvesting chromophores (stilbene and terphenylene–divinylene) and an electron acceptor (titanium oxide) is described. Efficient energy‐ and electron‐transfer processes may take place in these systems. Covalent bonding between the acceptor chromophores and the titania/silica matrix would be important for electron transfer, whereas fluorescence resonant energy transfer (FRET) would strongly depend on the ratio of donor to acceptor chromophores. Time‐resolved spectroscopy was employed to elucidate the detailed photophysical processes. The coupling of FRET and electron transfer was shown to work coherently to lead to photocurrent enhancement. The photocurrent responses reached a maximum when the hybrid‐material thin film contained 60 % acceptor and 40 % donor. 相似文献
9.
Heilemann M Tinnefeld P Sanchez Mosteiro G Garcia Parajo M Van Hulst NF Sauer M 《Journal of the American Chemical Society》2004,126(21):6514-6515
We demonstrate the synthesis and spectroscopic characterization of an unidirectional photonic wire based on four highly efficient fluorescence energy-transfer steps (FRET) between five spectrally different chromophores covalently attached to double-stranded DNA. The DNA-based modular conception enables the introduction of various chromophores at well-defined positions and arbitrary interchromophore distances. While ensemble fluorescence measurements show overall FRET efficiencies between 15 and 30%, single-molecule spectroscopy performed on four spectrally separated detectors easily uncovers subpopulations that exhibit overall FRET efficiencies of up to approximately 90% across a distance of 13.6 nm and a spectral range of approximately 200 nm. Fluorescence trajectories of individual photonic wires show five different fluorescence intensity patterns which can be ascribed to successive photobleaching events. 相似文献
10.
Sahoo H Roccatano D Hennig A Nau WM 《Journal of the American Chemical Society》2007,129(31):9762-9772
Fluorescence resonance energy transfer (FRET) from the amino acid tryptophan (Trp) as donor and a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as acceptor in peptides of the general structure Trp-(Pro)n-Dbo-NH2 (n = 1-6) was investigated by steady-state and time-resolved fluorescence, CD, and NMR spectroscopy as well as by molecular dynamics (MD) simulations (GROMOS96 force field). The Trp/Dbo FRET pair is characterized by a very short F?rster radius (R0 ca. 9 A), which allowed distance determinations in such short peptides. Water and propylene glycol were investigated as solvents. The peptides were designed to show an early nucleation of the poly(Pro)II (PPII) secondary helix structure for n > or = 2, which was confirmed by their CD spectra. The shortest peptide (n = 1) adopts preferentially the trans conformation about the Trp-Pro bond, as confirmed by NMR spectra. The FRET efficiencies ranged 2-72% and were found to depend sensitively on the peptide length, i.e., the number of intervening proline residues. The analysis of the FRET data at different levels of theory (assuming either a fixed distance or distance distributions according to a wormlike chain or Gaussian model) afforded donor-acceptor distances between ca. 8 A (n = 1) and ca. 16 A (n = 6) in water, which were found to be similar or slightly higher in propylene glycol. The distances afforded by the Trp/Dbo FRET pair were found to be reasonable in comparison to literature data, expectations from the PPII helix structure, and the results from MD simulations. The persistence lengths for the longer peptides were found to lie at 30-70 A in water and 220 +/- 40 A in propylene glycol, suggesting a more rigid PPII helical structure in propylene glycol. A detailed comparison with literature data on FRET in polyprolines demonstrates that the donor-acceptor distances extracted by FRET are correlated with the F?rster radii of the employed FRET pairs. This demonstrates the limitations of using FRET as a spectroscopic ruler for short polyprolines, which is presumably due to the breakdown of the point dipole approximation in F?rster theory, when the size of the chromophores becomes comparable or larger than the distances under investigation. 相似文献
11.
Cotlet M Vosch T Habuchi S Weil T Müllen K Hofkens J De Schryver F 《Journal of the American Chemical Society》2005,127(27):9760-9768
We report on the ensemble and single-molecule (SM) dynamics of F?rster resonance energy transfer (FRET) in a multichromophoric rigid polyphenylenic dendrimer (triad) with spectrally different rylene chromophores featuring distinct absorption and emission spectra which cover the whole visible spectral range: a terrylenediimide (TDI) core, four perylenemonoimides (PMIs) attached at the scaffold, and eight naphthalenemonoimides (NMIs) at the rim. For FRET from PMI to TDI taking place with an efficiency of 99.5%, single triad molecules optically excited at 490 nm show fluorescence exclusively from the TDI side in the beginning of their emission. On 360-nm excitation, NMI chromophores transfer their excitation energy either directly or in a stepwise fashion to the core TDI, the latter case involving scaffold-substituted PMIs as intermediate acceptors. Indeed, SM experiments on 360-nm excitation evidence highly efficient FRET from NMI chromophores to the TDI core since individual triad molecules show fluorescence exclusively either from TDI or from an intermediate (oxidized) species but never from PMI. Because PMI and TDI are chromophores with high fluorescence quantum yields and high resistance to photobleaching compared to NMI, 360-nm excitation of a single triad molecule leads to bleaching of NMI chromophores with no chance for PMI to be observed. The spatial positioning and the spectral properties of the chosen rylene chromophores make this multichromophoric system an efficient light collector, able to capture light over the whole visible spectral range and to transfer it finally to the core TDI, the latter releasing it as red fluorescence. 相似文献
12.
Hinze G Haase M Nolde F Müllen K Basché T 《The journal of physical chemistry. A》2005,109(30):6725-6729
We have investigated electronic excitation energy transfer in a specifically designed bichromophoric donor/acceptor dyad in which the donor (perylenediimide) and acceptor (terrylenediimide) are linked by a rigid heptaphenyl-spacer. Because of the choice of the bridge, which defines the distance and orientation of the two chromophores, donor as well as acceptor emission is observed. The significantly smaller photostability of the donor allows for time-resolved measurements of the acceptor emission at the single-molecule level with and without energy transfer from the donor. By analyzing the differences of the rise/decay profiles for both pathways, we could determine time constants of energy transfer with high accuracy for single dyads. The results show that the experimental approach presented here works even for situations in which the energy transfer times are smaller than the temporal resolution of the detection system. 相似文献
13.
Song HE Kirmaier C Schwartz JK Hindin E Yu L Bocian DF Lindsey JS Holten D 《The journal of physical chemistry. B》2006,110(39):19121-19130
Static and time-resolved optical measurements are reported for two cyclic hexameric porphyrin arrays and their self-assembled complexes with guest chromophores. The hexameric hosts contain zinc porphyrins and 0 or 3 free base (Fb) porphyrins (denoted Zn(6) or Zn(3)Fb(3), respectively). The guests are a tripyridyl arene (TP) and a dipyridyl-substituted free base porphyrin (DPFb), each of which coordinates to zinc porphyrins of a host via pyridyl-zinc dative bonding. Each architecture is designed to have an overall gradient of excited-state energies that affords excitation funneling within the host and ultimately to the guest. Collectively, the studies delineate the various pathways, mechanisms, and rate constants of energy flow among the weakly coupled constituents of the host-guest complexes. The pathways include downhill unidirectional energy transfer between adjacent chromophores, bidirectional energy migration between identical chromophores, and energy transfer between nonadjacent chromophores. The energy transfer to the lowest-energy chromophore(s) within the backbone of a hexameric host (Fb porphyrins in Zn(3)Fb(3) or pyridyl-coordinated zinc porphyrins in Zn(6)*TP and Zn(6)*DPFb) proceeds primarily via a through-bond mechanism; the transfer is rapid (approximately 40 ps depending on the array) and essentially quantitative (>or=98%). The energy transfer from a pyridyl-coordinated zinc porphyrin of the host to the Fb porphyrin guest in the Zn(6)*DPFb complex is almost exclusively F?rster through-space in nature; this process is much slower ( approximately 1 ns) and has a lower yield (65%). These studies highlight the utility of cyclic architectures for efficient light harvesting and energy transfer to a designated trapping site. 相似文献
14.
Finikova OS Troxler T Senes A DeGrado WF Hochstrasser RM Vinogradov SA 《The journal of physical chemistry. A》2007,111(30):6977-6990
Enhanced two-photon-absorbing (2PA) systems with triplet cores are currently under scrutiny for several biomedical applications, including photodynamic therapy (PDT) and two-photon microscopy of oxygen. The performance of so far developed molecules, however, is substantially below expected. In this study we take a detailed look at the processes occurring in these systems and propose ways to improve their performance. We focus on the interchromophore distance tuning as a means for optimization of two-photon sensors for oxygen. In these constructs, energy transfer from several 2PA chromophores is used to enhance the effective 2PA cross section of phosphorescent metalloporphyrins. Previous studies have indicated that intramolecular electron transfer (ET) can act as an effective quencher of phosphorescence, decreasing the overall sensor efficiency. We studied the interplay between 2PA, energy transfer, electron transfer, and phosphorescence emission using Rhodamine B-Pt tetrabenzoporphyrin (RhB-PtTBP) adducts as model compounds. 2PA cross sections (sigma2) of tetrabenzoporphyrins (TBPs) are in the range of several tens of GM units (near 800 nm), making TBPs superior 2PA chromophores compared to regular porphyrins (sigma2 values typically 1-2 GM). Relatively large 2PA cross sections of rhodamines (about 200 GM in 800-850 nm range) and their high photostabilities make them good candidates as 2PA antennae. Fluorescence of Rhodamine B (lambda(fl) = 590 nm, phi(fl) = 0.5 in EtOH) overlaps with the Q-band of phosphorescent PtTBP (lambda(abs) = 615 nm, epsilon = 98 000 M(-1) cm(-1), phi(p) approximately 0.1), suggesting that a significant amplification of the 2PA-induced phosphorescence via fluorescence resonance energy transfer (FRET) might occur. However, most of the excitation energy in RhB-PtTBP assemblies is consumed in several intramolecular ET processes. By installing rigid nonconducting decaproline spacers (Pro10) between RhB and PtTBP, the intramolecular ETs were suppressed, while the chromophores were kept within the F?rster r0 distance in order to maintain high FRET efficiency. The resulting assemblies exhibit linear amplification of their 2PA-induced phosphorescence upon increase in the number of 2PA antenna chromophores and show high oxygen sensitivity. We also have found that PtTBPs possess unexpectedly strong forbidden S0 --> T1 bands (lambda(max) = 762 nm, epsilon = 120 M-1 cm-1). The latter may overlap with the laser spectrum and lead to unwanted linear excitation. 相似文献
15.
We have applied fluorescence anisotropy and fluorescence resonance energy transfer (FRET) techniques to study the interaction between EcoRI DNA methyltransferase (M.EcoRI) and its target DNA in solution. Upon binding with M.EcoRI, the dsDNA containing GAATTC bends to flip out the second adenine for methylation. The binding affinity of M.EcoRI to two dsDNA fragments (20 and 38 bp) was studied with fluorescence anisotropy. Their binding constants at different temperatures from 20 to 40 degrees C were obtained, and the thermodynamic parameters of binding were derived. The results showed that M.EcoRI had a higher binding affinity to the short dsDNA strand than to the long one, and its binding to DNA was primarily entropy-driven. By labeling the 5' ends of the 20-bp dsDNA with two fluorescent dyes, fluorescein (FAM) and tetramethylrhodamine (TMR), we were able to monitor the enhanced TMR fluorescence in the presence of M.EcoRI. The end-to-end distance of the dsDNA determined from the FRET efficiency was changed from 72.4 to 63.4 A, and the DNA bending angle was estimated as 57.8 degrees . 相似文献
16.
Lewis FD Wu Y Zhang L Zuo X Hayes RT Wasielewski MR 《Journal of the American Chemical Society》2004,126(26):8206-8215
The synthesis, steady-state spectroscopy, and transient absorption spectroscopy of DNA conjugates possessing both stilbene electron donor and electron acceptor chromophores are described. These conjugates are proposed to form nicked DNA dumbbell structures in which a stilbenedicarboxamide acceptor and stilbenediether donor are separated by variable numbers of A-T or G-C base pairs. The nick is located either adjacent to one of the chromophores or between two of the bases. Thermal dissociation profiles indicate that stable structures are formed possessing as few as two A-T base pairs. Circular dichroism (CD) spectra in the base pair region are characteristic of B-DNA duplex structures, whereas CD spectra at longer wavelengths display two bands attributed to exciton coupling between the two stilbenes. The sign and intensity of these bands are dependent upon both the distance between the chromophores and the dihedral angle between their transition dipoles [Deltaepsilon approximately Rda(-2) sin(2theta)]. Pulsed laser excitation of the stilbenediamide results in creation of the acceptor-donor radical ion pair, which decays via charge recombination. The dynamics of charge separation and charge recombination display an exponential distance dependence, similar to that observed previously for systems in which guanine serves as the electron donor. Unlike exciton coupling between the stilbenes, there is no apparent dependence of the charge-transfer rates upon the dihedral angle between donor and acceptor stilbenes. The introduction of a single G-C base pair between the donor and acceptor results in a change in the mechanism for charge separation from single step superexchange to hole hopping. 相似文献
17.
Two different metrics are used to assess Forster resonance energy transfer (FRET) between fluorophores in the steady state: (i) acceptor-quenching of donor fluorescence E (also known as transfer efficiency) and (ii) donor-excited acceptor fluorescence F(A) (Dex). While E is still more widely used, F(A) (Dex) has been gaining in popularity for practical reasons among experimentalists who study biomembranes. Here, for the special case of membrane-bound fluorophores, we present a substantial body of experimental evidence that justifies the use of simple Stern-Volmer expressions when modeling either FRET metric under dilute-probe conditions. We have also discovered a dilute-regime correspondence between our Stern-Volmer expression for E and Wolber and Hudson's series approximation for steady-state Forster quenching in two dimensions (2D). This novel correspondence allows us to interpret each of our 2D quenching constants in terms of both (i) an effective Forster distance and (ii) two maximum acceptor-concentration limits, each of which defines its own useful experimental regime. Taken together, our results suggest a three-step strategy toward designing more effective steady-state FRET experiments for the study of biomembranes. 相似文献
18.
Specific sequential arrangements of three kinds of chromophores separated by regulated distances equaling approximately one pitch of the DNA duplex (34?Å) in non-covalent molecular assembly systems are constructed using chromophore/oligo-DNA conjugates. Vectorial photoenergy transmission along the DNA helix axis by fluorescence resonance energy transfer (FRET) in a sequential chromophore array is observed and analyzed by time-resolved fluorescence spectroscopy and lifetime measurements using a femtosecond pulse laser system. The results suggest a FRET occurs on a picosecond scale between the donor chromophore and the acceptor chromophore through a mediator chromophore via a multi-step FRET over the molecular assemblies (two helical pitches, 68?Å). 相似文献
19.
Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins. 总被引:1,自引:0,他引:1
Mark A Rizzo Gerald Springer Katsuhisa Segawa Warren R Zipfel David W Piston 《Microscopy and microanalysis》2006,12(3):238-254
Detection of F?rster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater F?rster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET. 相似文献
20.
Chuyun Deng 《Talanta》2010,82(2):771-882
Fluorescence resonance energy transfer (FRET) is widely used to obtain the distance between a donor and an acceptor in biological research. However, the detection of FRET efficiencies with fluorescence microscopy imaging systems remains a great challenge due to the difficulties of transferring gray scales of the images into fluorescence intensities, and the absence of exact quantum yields of donors and acceptors. Herein, we presented a new method to detect the FRET efficiency in imaging systems by analyzing the photo-bleaching-induced changes in fluorescent intensities of quantum dots (QDs, donors) and Cy5 dyes (acceptors). Our method is different from the previous acceptor-photo-bleaching studies in imaging systems by theoretically analyzing the bleaching process, and bringing forward a new parameter which is universal for samples of the same kind. It is convenient for calculating FRET efficiencies. There is hardly any spectral crosstalk between 605QD and Cy5, thus the FRET result is more accurate than that of many other common FRET pairs. The lengths of single-stranded and double-stranded DNA fragments in solution were determined via the analysis of FRET efficiency values. This technique provides a reliable approach to study biomacromolecules in living cells through fluorescent imaging and in situ measurements. 相似文献