S' subsite mapping of serine proteases based on fluorescence resonance energy transfer.

A microassay based on fluorescence resonance energy transfer has been developed to determine the S' specificity of serine proteases. The protease-catalyzed acyl transfer from a fluorescing acyl donor ester to a P'1/P'2 variable hexapeptide library of nucleophiles labeled with a fluorescence quencher leads to an internally quenched peptide product and a fluorescent hydrolysis product. The amount of fluorescence quenching allows one to draw conclusions about the interaction of the nucleophile at the S' sites of the protease. o-Aminobenzoic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The P'1/P'2 variable hexapeptide library with the general structure H-Xaa-Ala-Ala-Ala-Tyr(NO2)-Gly-OH and H-Ala-Xaa-Ala-Ala-Tyr(NO2)-Gly-OH, where Xaa represents Arg, Lys, Met, Phe, Ala, Gly, Ser, Gln and Glu, was prepared by solid-phase synthesis. Investigations of the S' specificity of trypsin, chymotrypsin and trypsin variants show that this assay is a fast and sensitive screening method for S' subsite mapping of serine proteases and is suitable for a high throughput screening. The assay might be useful for the development of restriction proteases and the estimation of yields in enzymatic peptide synthesis.     

Energy transfer and charge separation kinetics in photosystem I: Part 1: Picosecond transient absorption and fluorescence study of cyanobacterial photosystem I particles

The energy transfer and charge separation kinetics of a photosystem I (PS I) core particle of an antenna size of 100 chlorophyll/P700 has been studied by combined fluorescence and transient absorption kinetics with picosecond resolution. This is the first combined picosecond study of transient absorption and fluorescence carried out on a PS I particle and the results are consistent with each other. The data were analyzed by both global lifetime and global target analysis procedures. In fluorescence major lifetime components were found to be 12 and 36 ps. The shorter-lived one shows a negative amplitude at long wavelengths and is attributed to an energy transfer process between pigments in the main antenna Chl pool and a small long-wavelength Chl pool emitting around 720 nm whereas the longer-lived component is assigned to the overall charge separation lifetime. The lifetimes resolved in transient absorption are 7-8 ps, 33 ps, and [unk]1 ns. The shortest-lived one is assigned to energy transfer between the same pigment pools as observed also in fluorescence kinetics, the middle component of 33 ps to the overall charge separation, and the long-lived component to the lifetime of the oxidized primary donor P700⁺. The transient absorption data indicate an even faster, but kinetically unresolved energy transfer component in the main Chl pool with a lifetime <3 ps. Several kinetic models were tested on both the fluorescence and the picosecond absorption data by global target analysis procedures. A model where the long-wave pigments are spatially and kinetically connected with the reaction center P700 is favored over a model where P700 is connected more closely with the main Chl pool. Our data show that the charge separation kinetics in these PS I particles is essentially trap limited. The relevance of our data with respect to other time-resolved studies on PS I core particles is discussed, in particular with respect to the nature and function of the long-wave pigments. From the transient absorption data we do not see any evidence for the occurrence of a reduced Chl primary electron acceptor, but we also can not exclude that possibility, provided that reoxidation of that acceptor should occur within a time <40 ps.     

Voltage sensing by fluorescence resonance energy transfer in single cells.

A new mechanism has been developed for achieving fast ratiometric voltage-sensitive fluorescence changes in single cells using fluorescence resonance energy transfer. The mechanism is based on hydrophobic fluorescent anions that rapidly redistribute from one face of the plasma membrane to the other according to the Nernst equation. A voltage-sensitive fluorescent readout is created by labeling the extracellular surface of the cell with a second fluorophore, here a fluorescently labeled lectin, that can undergo energy transfer with the membrane-bound sensor. Fluorescence resonance energy transfer between the two fluorophores is disrupted when the membrane potential is depolarized, because the anion is pulled to the intracellular surface of the plasma membrane far from the lectin. Bis-(1,3-dialkyl-2-thiobarbiturate)-trimethineoxonols, where alkyl is n-hexyl and n-decyl (DiSBA-C6-(3) and DiSBA-C10-(3), respectively) can function as donors to Texas Red labeled wheat germ agglutinin (TR-WGA) and acceptors from fluorescein-labeled lectin (FI-WGA). In voltage-clamped fibroblasts, the translocation of these oxonols is measured as a displacement current with a time constant of approximately 2 ms for 100 mV depolarization at 20 degrees C, which equals the speed of the fluorescence changes. Fluorescence ratio changes of between 4% and 34% were observed for a 100-mV depolarization in fibroblasts, astrocytoma cells, beating cardiac myocytes, and B104 neuroblastoma cells. The large fluorescence changes allow high-speed confocal imaging.     

Excitation energy transfer in the light-harvesting chlorophyll a/b.protein

The "light-harvesting chlorophyll a/b.protein" described by Thornber has been prepared electrophoretically from spinach chloroplasts. The optical properties relevant to energy transfer have been measured in the red region (i.e. 600-700 nm). Measurements of the absorption spectrum, fluorescence excitation spectrum and excitation dependence of the fluorescence emission spectrum of this protein confirm that energy transfer from chlorophyll b to chlorophyll a is highly efficient, as is the case in concentrated chlorophyll solutions and in vivo. The excitiation dependence of the fluorescence polarization shows a minimum polarization of 1.9% at 650 nm which is the absorption maximum of chlorophyll b in the protein and rises steadily to a maximum value of 13.8% at 695 nm, the red edge of the chlorophyll a absorption band. Analysis of these measurements shows that at least two unresolved components must be responsible for the chlorophyll a absorption maximum. Comparison of polarization measurements with those observed in vivo shows that most of the depolarization observed in vivo can take place within a single protein. Circular dichroism measurements show a double structure in the chlorophyll b absorption band which suggest an exciton splitting not resolved in absorption. Analysis of these data yields information about the relative orientation of the So leads to S1 transition moments of the chlorophyll molecules within the protein.     

Simultaneous DNA binding, bending, and base flipping: evidence for a novel M.EcoRI methyltransferase-DNA complex

We measured the kinetics of DNA bending by M.EcoRI using DNA labeled at both 5'-ends and observed changes in fluorescence resonance energy transfer. Although known to bend its cognate DNA site, energy transfer is decreased upon enzyme binding. This unanticipated effect is shown to be robust because we observe the identical decrease with different dye pairs, when the dye pairs are placed on the respective 3'-ends, the effect is cofactor- and protein-dependent, and the effect is observed with duplexes ranging from 14 through 17 base pairs. The same labeled DNA shows the anticipated increased energy transfer with EcoRV endonuclease, which also bends this sequence, and no change in energy transfer with EcoRI endonuclease, which leaves this sequence unbent. We interpret these results as evidence for an increased end-to-end distance resulting from M.EcoRI binding, mediated by a mechanism novel for DNA methyltransferases, combining DNA bending and an overall expansion of the DNA duplex. The M.EcoRI protein sequence is poorly accommodated into well defined classes of DNA methyltransferases, both at the level of individual motifs and overall alignment. Interestingly, M.EcoRI has an intercalation motif observed in the FPG DNA glycosylase family of repair enzymes. Enzyme-dependent changes in anisotropy and fluorescence resonance energy transfer have similar rate constants, which are similar to the previously determined rate constant for base flipping; thus, the three processes are nearly coincidental. Similar fluorescence resonance energy transfer experiments following AdoMet-dependent catalysis show that the unbending transition determines the steady state product release kinetics.     

The estimation of distances between specific backbone-labeled sites in DNA using fluorescence resonance energy transfer.

A series DNA helices of twenty-four base pairs has been prepared for the study of fluorescence resonance energy transfer. Each of the DNA helices contains two phosphorothioate diesters (one in each strand) at pre-selected sites for introduction of the desired donor and acceptor fluorophores. The phosphorothioate-containing oligodeoxynucleotides have been prepared as pure Rp or Sp derivatives or as deastereomeric mixtures. Fluorescein and eosin are employed as the respective donor and acceptor fluorophores. A series of donor-acceptor pairs was generated by labeling of the appropriate phosphorothioate diester with the desired fluorophore and annealing the two complementary DNA strands (one containing the acceptor and one containing the donor fluorophore) to form the double-stranded helix. The 24-mer helices containing two covalently attached fluorophores exhibited some thermal destabilization and the extent of this destabilization was dependent upon the stereochemical orientation of the fluorophore. The Sp derivatives direct the fluorophore out, away from the the DNA helix, while the Rp derivatives direct the fluorophore toward the major groove. As expected, the Sp labeled duplexes were more stable than the corresponding Rp labeled sequences. However, all of the duplex structures formed were stable under the conditions used to measure energy transfer. Energy transfer could be observed with these complexes from the quenching of the donor fluorescence in the presence of the acceptor fluorophore. Using F?rster's theories, distances separating the fluorophores could be calculated that were generally in reasonable agreement with the distances expected in an idealized B-form DNA helix. However anomalous results were obtained for one donor/acceptor pair where the expected distance was less than 20 A. Fluorescence anisotropy values determined in solutions of varying viscosity were quite high suggesting that the fluorophores did not experience complete freedom of movement when attached to the DNA helix.     

Time-resolved detection of energy transfer: theory and application to immunoassays

Energy-transfer measurements based upon acceptor fluorophore emission are plagued with background fluorescence resulting from absorption of the excitation light by the acceptor fluorophore. The present work examines the use of a long-lifetime donor fluorophore and a short-lifetime acceptor fluorophore, combined with pulsed-laser excitation and electronic gating of detector signals, to separate the component of acceptor emission due to energy transfer from the component due to absorption of the excitation light. Theoretical equations describing the acceptor fluorescence and integrated acceptor fluorescence show that increasing the integration delay relative to the excitation pulse should greatly enhance detection of the energy-transfer component. The time-resolved detection of energy transfer was tested in a competitive immunoassay format in which antibodies to human immunoglobulin G (IgG) F(ab')2 fragments were covalently labeled with pyrenebutyrate (tau = 100 ns) and IgG Fab' fragments were covalently labeled with B-phycoerythrin (tau = 2.5 ns). Solutions containing these two conjugates exhibited energy transfer from the pyrenebutyrate to the B-phycoerythrin upon excitation with a nitrogen laser. Acceptor emission was measured with 0- and 20-ns integration delays and the ratios of the energy-transfer component to the laser-excited component were found to increase by 9- to 15-fold when the 20-ns delay was used in three series of immunoassays. Good agreement between the experimental data and theory was obtained following convolution of the theoretical fluorescence responses with the instrumental response of the fluorometer.     

Effect of phospholipid:protein ratio on the state of aggregation of the (Ca2+-Mg2+)-ATPase

The organization of the (Ca2+-Mg2+)-ATPase has been studied in reconstituted systems by fluorescence polarization of the ATPase labeled with fluorescein isothiocyanate (FITC) and resonance energy transfer between ATPase labeled with FITC and with eosin isothiocyanate (EITC). The fluorescence polarization of FITC-ATPase was found to decrease with increasing labeling ratio FITC:ATPase, indicating depolarization as a result of resonance energy transfer between ATPase molecules. Fluorescence polarization was, however, independent of the molar ratio of phospholipid to protein above a molar ratio of 50:1. Resonance energy transfer between FITC-ATPase and EITC-ATPase was also found to be independent of phospholipid:protein ratio. It is suggested therefore that the ATPase is not randomly distributed in the plane of the membrane but rather forms ordered clusters (probably rows of monomers or dimers) on the fluorescence time scale (nanoseconds) even in the presence of a large excess of phospholipid. This organization within the membrane is dependent both on the chemical structure of the phospholipid and on its physical phase.     

Fluorescence resonance energy transfer between specific-labeled sites on DNA.

Fluorescence resonance energy transfer on DNA has been studied for the estimation of distances between specific sites. Two kind of fluorophores, donor and acceptor, were incorporated on double-stranded DNA via phosphorothioate linkage (Sp, Rp, or racemic mixture). The thermal stability of labeled DNA's was slightly dependent on the stereochemical orientation of fluorophore, however all of the duplex structures were stable under the conditions for fluorescence study. The distances between donor and acceptor fluorophores, estimated from fluorescence energy transfer, generally agreed with the expected distance in a B-type DNA for the limiting distance.     

Quantification of Protein-Lipid Selectivity using FRET: Application to the M13 Major Coat Protein

Quantification of lipid selectivity by membrane proteins has been previously addressed mainly from electron spin resonance studies. We present here a new methodology for quantification of protein-lipid selectivity based on fluorescence resonance energy transfer. A mutant of M13 major coat protein was labeled with 7-diethylamino-3((4'iodoacetyl)amino)phenyl-4-methylcoumarin to be used as the donor in energy transfer studies. Phospholipids labeled with N-(7-nitro-2-1,3-benzoxadiazol-4-yl) were selected as the acceptors. The dependence of protein-lipid selectivity on both hydrophobic mismatch and headgroup family was determined. M13 major coat protein exhibited larger selectivity toward phospholipids which allow for a better hydrophobic matching. Increased selectivity was also observed for anionic phospholipids and the relative association constants agreed with the ones already presented in the literature and obtained through electron spin resonance studies. This result led us to conclude that fluorescence resonance energy transfer is a promising methodology in protein-lipid selectivity studies.     

Interactions of the dimethyldiazaperopyrenium dication with nucleic acids. 2. Binding to double-stranded polynucleotides

The interactions of dimethyldiazaperopyrenium dication (1) with DNA have been studied by spectroscopic methods: absorption, static and dynamic fluorescence, and linear dichroism. 1 binds strongly to DNA at 250 mM NaCl, with a higher affinity for G-C pairs as compared to A-T pairs. The dye fluorescence is enhanced when it is bound to A-T pairs, whereas the emission is quenched in the vicinity of G-C pairs. Evidence for intercalation has been obtained via energy transfer and linear dichroism measurements.     

Study on the fluorescent enhancement effect in terbium-gadolinium-protein-sodium dodecyl benzene sulfonate system and its application on sensitive detection of protein at nanogram level

The co-luminescence effect in a terbium-gadolinium-protein-sodium dodecyl benzene sulfonate (SDBS) system is reported here. Based on it, the sensitive quantitative analysis of protein at nanogram levels is established. The co-luminescence mechanism is studied using fluorescence, resonance light scattering (RLS), absorption spectroscopy and NMR measurement. It is considered that protein could be unfolded by SDBS, then a efficacious intramolecular fluorescent energy transfer occurs from unfolded protein to rare earth ions through SDBS acting as a "transfer bridge" to enhance the emission fluorescence of Tb3+ in this ternary complex of Tb-SDBS-BSA, where energy transfer from protein to SDBS by aromatic ring stacking is the most important step. Cooperating with the intramolecular energy transfer above is the intermolecular energy transfer between the simultaneous existing complexes of both Tb3+ and Gd3+. The fluorescence quantum yield is increased by an energy-insulating sheath, which is considered to be another reason for the resulting enhancement of the fluorescence. F?rster theory is used to calculate the distribution of enhancing factors and has led to a greater understanding of the mechanisms of energy transfer.     

Quantitative calculations of fluorescence polarization and absorption anisotropy kinetics of double- and triple-chromophore complexes with energy transfer.

A new method is presented for calculation of the fluorescence depolarization and kinetics of absorption anisotropy for molecular complexes with a limited number of chromophores. The method considers absorption and emission of light by both chromophores, and also energy transfer between them, with regard to their mutual orientations. The chromophores in each individual complex are rigidly positioned. The complexes are randomly distributed and oriented in space, and there is no energy transfer between them. The new "practical" formula for absorption anisotropy and fluorescence depolarization kinetics, P(t) = [3B(t) - 1 + 2A(t)]/[3 + B(t) + 4A(t)], is derived both for double- and triple-chromophore complexes with delta-pulse excitation. The parameter B(t) is given by (a) B(t) = cos2(theta) for double-chromophore complexes, and (b) B(t) = q12(t)cos2(theta 12) + q13(t)-cos2(theta 13) + q23(t)cos2(theta 23) for triple-chromophore complexes, where q12(t) + q13(t) + q23(t) = 1. Here theta ij are the angles between the chromophore transition dipole moments in the individual molecular complex. The parameters qij(t) and A(t) are dependent on chromophore spectroscopic features and on the rates of energy transfer.     

Non-linear least-squares methods applied to the analysis of fluorescence energy transfer measurements.

We describe a new method for calculating the efficiency of fluorescence energy transfer on labeled macromolecules using steady-state measurements. A single estimation of the efficiency value is obtained by a global analysis of all the measurement data sets (absorption, emission and excitation spectra) using non-linear least-squares. The method was tested on simulated and experimental data obtained from three simple model compounds: an equimolar mixture of tryptophan-tyrosine and two peptides, Trp-Tyr and Trp-Gly-Gly-Tyr, in which transfer efficiencies are respectively nearly 100% and 50%. The method was found to be reliable and provides methodological and quantitative advantages in regard to the sequential methods currently used.     

Fluorescent tandem phycobiliprotein conjugates. Emission wavelength shifting by energy transfer.

A fluorescent tandem phycobiliprotein conjugate with a large Stokes shift was prepared by the covalent attachment of phycoerythrin to allophycocyanin. The efficiency of energy transfer from phycoerythrin to allophycocyanin in this disulfide-linked conjugate was 90%. A distinctive feature of this phycocyanin conjugate is the wide separation between the intense absorption maximum of phycoerythrin (epsilon = 2.4 x 10(6) cm-1 M-1 at 545 nm) and the fluorescence emission maximum of allophycocyanin (660 nm). Energy transfer from a donor to a covalently attached acceptor can be used to adjust the magnitude of the Stokes shift. Tandem phycobiliprotein conjugates can be used to advantage in fluorescence-activated cell sorting, fluorescence microscopy, and fluorescence immunoassay analyses.     

Photodichroic studies of the photoreaction center from Rhodospirillum Rubrum. I. Attribution of P870 to two non parallel dipoles

A randomly oriented sample of photoreaction center prepared from Rhodospirillum rubrum was excited at 77 °K by an actinic linearly polarized light of 870 nm. Under such conditions, only those chromophores with components of their absorption dipoles oriented parallel to the polarization of the actinic light are bleached. The change in absorbance at 900 nm of this photoselected sample was observed while varying the angle of polarization of a weak measuring light. The polarization of the absorbance change was thus evaluated as 0.25.

This value is interpreted to mean that P₈₇₀ is attributable to two absorption dipoles forming an angle included between 35.75° and 90°. Comparison with the p value of 0.5 obtained on a similar preparation by polarization of fluorescence (Ebrey, T. G. and Clayton, R. K. (1969) Photochem. Photobiol. 10, 109–117) leads to the conclusion that either these two dipoles emit fluorescence without being coupled by singlet-singlet energy transfer or that only one of them is a fluorescence emitter in the absence of reversible singlet-singlet energy transfer.     

Spectroscopic properties of the main-form and high-salt peridinin-chlorophyll a proteins from Amphidinium carterae

The main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a proteins from the dinoflagellate Amphidinium carterae were investigated using absorption, fluorescence, fluorescence excitation, two-photon, and fast-transient optical spectroscopy. Pigment analysis has demonstrated previously that MFPCP contains eight peridinins and two chlorophyll (Chl) a molecules, whereas HSPCP has six peridinins and two Chl a molecules [Sharples, F. P., et al. (1996) Biochim. Biophys. Acta 1276, 117-123]. Absorption spectra of the complexes were recorded at 10 K and analyzed in the 400-600 nm region by summing the individual 10 K spectra of Chl a and peridinin recorded in 2-MTHF. The absorption spectral profiles of the complexes in the Q(y) region between 650 and 700 nm were fit using Gaussian functions. The absorption and fluorescence spectra from both complexes exhibit several distinguishing features that become evident only at cryogenic temperatures. In particular, at low temperatures the Q(y) transitions of the Chls bound in the HSPCP complex are split into two well-resolved bands. Fluorescence excitation spectroscopy has revealed that the peridinin-to-Chl a energy transfer efficiency is high (>95%). Transient absorption spectroscopy has been used to measure the rate of energy transfer between the two bound Chls which is a factor of 2.9 slower in HSPCP than in MFPCP. The kinetic data are interpreted in terms of the F?rster mechanism describing energy transfer between weakly coupled, spatially fixed, donor-acceptor Chl a molecules. The study provides insight into the molecular factors that control energy transfer in this class of light-harvesting pigment-protein complexes.     

Relaxation time, interthiol distance, and mechanism of action of ribosomal protein S1

The two sulfhydryl groups of ribosomal protein S1 from Escherichia coli have been labeled with fluorescent maleimides and the distance between them has been determined by nonradiative energy transfer. This distance was found to be approximately 27 A for both free S1 and S1 bound to 30 S subunits. This value probably represents an upper limit. The position of the fluorescence emission maximum indicates that both sulfhydryl groups are in a relatively hydrophobic environment. When poly(U) is added to labeled S1, either free or in 30 S subunits, the emission maximum shifts to the red by about 3 nm but without a detectable change in the interthiol distance. S1 labeled at one or both of its sulfhydryl groups retains most of its ability to enhance poly(U)-directed polyphenylalanine synthesis. About the same concentration of poly(U) is required to give the maximum shift in fluorescence as is required to give maximum polyphenylalanine synthesis, indicating that S1 binds poly(U) during translation. The peptide initiation inhibitor aurintricarboxylic acid almost completely quenches the fluorescence from either labeled sulfhydryl groups in S1 bound to ribosomes or free in solution. This quenching probably is due to energy transfer from the labeled sulfhydryls to bound aurintricarboxylic acid. Fluorescence anisotropy measurements indicated that the C-terminal domain of S1 is relatively rigid, but retains some independent movement when attached to ribosomes. The overall data are consistent with a model in which a region near the two sulfhydryl groups in the elongated C-terminal domain functions to sequester and bind mRNA to the ribosome during peptide synthesis.     

Study on the mechanism of the interaction between acteoside and pepsin using spectroscopic techniques

The interaction of acteoside with pepsin has been investigated using fluorescence spectra, UV/vis absorption spectra, three‐dimensional (3D) fluorescence spectra and synchronous fluorescence spectra, along with a molecular docking method. The fluorescence experiments indicate that acteoside can quench the intrinsic fluorescence of pepsin through combined quenching at a low concentration of acteoside, and static quenching at high concentrations. Thermodynamic analysis suggests that hydrogen bonds and van der Waal's forces are the main forces between pepsin and acteoside. According to the theory of Förster's non‐radiation energy transfer, the binding distance between pepsin and acteoside was calculated to be 2.018 nm, which implies that energy transfer occurs between acteoside and pepsin. In addition, experimental results from UV/vis absorption spectra, 3D fluorescence spectra and synchronous fluorescence spectra imply that pepsin undergoes a conformation change when it interacts with acteoside. Copyright © 2015 John Wiley & Sons, Ltd.