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1.
The properties of the tyrosine and tyrosinate emissions from brain S-100b have been studied by nanosecond time-resolved fluorescence at emission wavelengths in the range 305 to 365 nm. The effect of pH on the fluorescence has been studied at pH 6.5, 7.5, and 8.5 for the Ca(II) apo and holo forms of the protein, and for the apo and holo forms in the presence and absence of Zn(II) at pH 7.5. The fluorescence decay is biexponential at pH 8.5 and triexponential at pH 6.5 and 7.5. The three components of the decay have wavelength and metal ion dependent lifetimes in the ranges 0.06 to 1.05 ns, 0.49 to 3.76 ns, and 3.60 to 14.5 ns. The observation of a long lifetime component at wavelengths characteristic of emission from tyrosinate suggests that in class A proteins this may be a useful diagnostic of the environment of tyrosine in their native structures. The time-resolved emission spectra provide evidence for efficient, subnanosecond protolysis of the excited state of the single tyrosine (Tyr17) under all conditions studied except in 6 M guanidium chloride in which the protein shows only emission from tyrosine (lambda em 305 nm), suggesting that the tyrosinate emission is a property of the tertiary structure of the native protein. The Zn(II)-dependence of the fluorescence is fully consistent with the earlier suggestion that Tyr17 is near the Zn(II) binding site and remote from the high affinity Ca(II) binding site. 相似文献
2.
Tryptophanyl fluorescence lifetime distribution of hyperthermophilic beta-glycosidase from molecular dynamics simulation: a comparison with the experimental data
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Bismuto E Martelli PL Casadio R Irace G 《Protein science : a publication of the Protein Society》2000,9(9):1730-1742
A molecular dynamics simulation approach has been utilized to understand the unusual fluorescence emission decay observed for beta-glycosidase from the hyperthermophilic bacterium Solfolobus sulfotaricus (Sbeta gly), a tetrameric enzyme containing 17 tryptophanyl residues for each subunit. The tryptophanyl emission decay of Sbeta gly results from a bimodal distribution of fluorescence lifetimes with a short-lived component centered at 2.5 ns and a long-lived one at 7.4 ns (Bismuto E, Nucci R, Rossi M, Irace G, 1999, Proteins 27:71-79). From the examination of the trajectories of the side chains capable of causing intramolecular quenching for each tryptophan microenvironment and using a modified Stern-Volmer model for the emission quenching processes, we calculated the fluorescence lifetime for each tryptophanyl residue of Sbeta gly at two different temperatures, i.e., 300 and 365 K. The highest temperature was chosen because in this condition Sbeta gly evidences a maximum in its catalytic activity and is stable for a very long time. The calculated lifetime distributions overlap those experimentally determined. Moreover, the majority of trytptophanyl residues having longer lifetimes correspond to those originally identified by inspection of the crystallographic structure. The tryptophanyl lifetimes appear to be a complex function of several variables, such as microenvironment viscosity, solvent accessibility, the chemical structure of quencher side chains, and side-chain dynamics. The lifetime calculation by MD simulation can be used to validate a predicted structure by comparing the theoretical data with the experimental fluorescence decay results. 相似文献
3.
Picosecond-resolved fluorescence spectra of D-amino-acid oxidase. A new fluorescent species of the coenzyme 总被引:1,自引:0,他引:1
Protein dynamics of D-amino-acid oxidase in the picosecond region was investigated by measuring time-resolved fluorescence of the bound coenzyme, FAD. The observed nonexponential fluorescence decay curves were analyzed with four-exponential decay functions. The fluorescence lifetimes at the best fit were 26.6 +/- 0.7 ps, 44.0 +/- 4.2 ps, 177 +/- 11 ps, and 2.28 +/- 0.21 ns at 20 degrees C and 25.2 +/- 3.0 ps, 50.3 +/- 8.7 ps, 228 +/- 27 ps, and 2.75 +/- 0.33 ns at 5 degrees C. Component fractions with the shortest lifetime, ca. 26 ps, were always negative and close to -1. The other fluorescent components of the lifetimes, ca. 47 ps, 200 ps, and 2.6 ns, with positive fractions were assigned to different forms of the enzyme including the dimer, the monomer, and free FAD dissociated from the enzyme. Measurements of the time-resolved fluorescence spectra revealed that the maximum wavelengths of the spectra shifted toward shorter wavelength by 65 nm at 20 degrees C and 36 nm at 5 degrees C within 100 ps after pulsed excitation. The remarkable blue shift was not observed in free FAD. The first spectra immediately after the excitation of the enzyme exhibited maximum wavelengths of 584 nm at 20 degrees C and 557 nm at 5 degrees C. The fluorescence spectra obtained at times later than 100 ps are in good agreement with the one obtained under steady-state excitation of D-amino-acid oxidase.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p-terphenyl, p-bis[2-(5-phenyloxazolyl)]benzene and N-acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p-terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p-terphenyl (20 degrees C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile-up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile-up effects play a role at this frequency. The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple-exponential fluorescence decay. The anisotropy decay of albumin was found to be mono-exponential with a rotational correlation time of 26 ns (20 degrees C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for lambda ex = 295 nm and 36 ns for lambda ex = 300 nm at 20 degrees C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple-exponential fluorescence decay. A single-exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion. 相似文献
5.
The tryptophanyl emission decay of beta-glycosidase from the extremophilic archaeon Sulfolobus solfataricus (Sbetagly) has been investigated by frequency domain fluorometry. The data were analyzed in terms of sum of discrete lifetimes as well as in terms of quasi- continuous lifetime distributions of different shape. At neutral pH the emission decay is characterized by two components: a long-lived component, centered at 7.4 ns, and a short one at 2.7 ns, irrespective of the decay scheme used for the interpretation of the experimental results. The effects of an irreversible inhibitor, that is, cyclophellitol, and that of a powerful denaturant such as guanidinium hydrochloride on the dynamics of Sbetagly has been investigated by observing the changes induced in the two components of the tryptophanyl emission decay. The addition of cyclophellitol to native Sbetagly reduces the contribution of the short-lived component but does not affect the long-lived one. Increasing concentrations of guanidinium hydrochloride differently affect the contributions of the two emission components. Higher concentrations were required to unfold the molecular regions containing the long-lived indolic fluorophores. These results indicate that the long-lived contribution arises from tryptophanyl residues deeply clustered in the interior of the protein matrix, whereas the short-lived one includes residues located in less rigid and more solvent accessible regions, some of which might be located in functionally important parts of protein. The knowledge of the crystallographic structure of Sbetagly allowed us to evaluate some average parameters for each tryptophanyl microenvironment in the Sbetagly such as hydrophobicity, structural flexibility, and ability of side chains to act as fluorescence quenchers. These results permitted to divide the tryptophanyl fluorescence of Sbetagly in the contribution of two emitting groups: one consisting of eight closely clustered tryptophans, that is, Trp 33, 36, 60, 84, 151 174, 425, and 433, responsible for the long-lived emission component and the other one, composed of nine tryptophans nearer to the subunit surface, that is, Trp 12, 156, 192, 287, 288, 316, 361, 376, 455, associable to the short-lived emission component. Finally, the examination of the tryptophanyl emission decay of the mesophilic beta-galactosidase from Escherichia coli (Cbetagal) and the Arrhenius analysis of its dependence on temperature indicated that the tryptophanyl environments of the mesophilic enzyme are rather homogeneous in consequence of a larger protein dynamics. 相似文献
6.
Zdenek Petrásek Franz-Josef Schmitt Christoph Theiss Joachim Huyer Min Chen Anthony Larkum Hans Joachim Eichler Klaus Kemnitz Hann-J?rg Eckert 《Photochemical & photobiological sciences》2005,4(12):1016-1022
The fluorescence decay spectra and the excitation energy transfer from the phycobiliproteins (PBP) to the chlorophyll-antennae of intact cells of the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina were investigated at 298 and 77 K by time- and wavelength-correlated single photon counting fluorescence spectroscopy. At 298 K it was found that (i) the fluorescence dynamics in A. marina is characterized by two emission peaks located at about 650 and 725 nm, (ii) the intensity of the 650 nm fluorescence depends strongly on the excitation wavelength, being high upon excitation of phycobiliprotein (PBP) at 632 nm but virtually absent upon excitation of chlorophyll at 430 nm, (iii) the 650 nm fluorescence band decayed predominantly with a lifetime of 70 +/- 20 ps, (iv) the 725 nm fluorescence, which was observed independent of the excitation wavelength, can be described by a three-exponential decay kinetics with lifetimes depending on the open or the closed state (F(0) or F(m)) of the reaction centre of Photosystem II (PS II). Based on the results of this study, it is inferred that the excitation energy transfer from phycobiliproteins to Chl d of PS II in A. marina occurs with a time constant of about 70 ps, which is about three times faster than the energy transfer from the phycobilisomes to PS II in the Chl a-containing cyanobacterium Synechococcus 6301. A similar fast PBP to Chl d excitation energy transfer was also observed at 77 K. At 77 K a small long-lived fluorescence decay component with a lifetime of 14 ns was observed in the 640-700 nm spectral range. However, it has a rather featureless spectrum, not typical for Chl a, and was only observed upon excitation at 400 nm but not upon excitation at 632 and 654 nm. Thus, this long-lived fluorescence component cannot be used as an indicator that the primary PS II donor of Acaryochloris marina contains Chl a. 相似文献
7.
The direct time-resolved fluorescence anisotropy of the single tryptophan residue in the polypeptide hormone adrenocorticotropin-(1-24) (ACTH) and the fluorescence decay kinetics of this residue (Trp-9) are reported. Two rotational correlation times are observed. One, occurring on the subnanosecond time scale, reflects the rotation of the indole ring, and the other, which extends into the nanosecond range, is dominated by the complex motions of the polypeptide chain. The fluorescence lifetimes of the single tryptophan in glucagon (Trp-25) and the 23-26 glucagon peptide were also measured. In all cases the fluorescence kinetics were satisfied by a double-exponential decay law. The fluorescence lifetimes of several tryptophan and indole derivatives and two tryptophan dipeptides were examined in order to interpret the kinetics. In close agreement with the findings of Szabo and Rayner [Szabo, A. G., & Rayner, D. M. (1980) J. Am. Chem. Soc. 102, 554-563], the tryptophan zwitterion exhibits emission wavelength dependent double-exponential decay kinetics. At 320 nm tau 1 = 3.2 ns and tau 2 = 0.8 ns, with alpha 1 = 0.7 and alpha 2 = 0.3. Above 380 nm only the 3.2-ns component is observed. By contrast the neutral derivative N-acetyltryptophanamide has a single exponential decay of 3.0 ns. The multiexponential decay kinetics of the polypeptides are discussed in terms of flexibility of the polypeptide chain and neighboring side-chain interactions. 相似文献
8.
The effect of membrane curvature on the fluorescence decay of 2-p-toluidinyl-naphthalene-6-sulfonic acid (TNS), 2-(9-anthroyloxy) stearic acid (2-AS) and 12-(9-anthroyloxy)-stearic acid (12-AS) was investigated for egg lecithin vesicles of average diameter dm = 22 nm and 250 nm. The biexponential fluorescence decay of TNS at the red edge of the emission spectrum was analysed according to the model of Gonzalo and Montoro [1]. Over the entire temperature range (1-40 degrees C) the small TNS labelled vesicles showed significantly shorter solvent relaxation times tau(r) than their larger counterparts (e.g. 1.3 ns compared with 2.1 ns at 5 degrees C), indicating a higher mobility of the hydrated headgroups in the highly curved, small vesicles. The fluorescence decay of both AS derivatives is also biexponential. While the shorter decay times (1-3 ns) are practically identical for small and large vesicles, the longer decay times (5-14 ns) are identical only for 12-AS but not for 2-AS. This indicates that the microenvironment is similar in small and large vesicles deep in the membrane in spite of the differences in curvature. 相似文献
9.
The peptide bond quenches tryptophan fluorescence by excited-state electron transfer, which probably accounts for most of the variation in fluorescence intensity of peptides and proteins. A series of seven peptides was designed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structure and side-chain chi(1) rotamer populations of the peptides were determined by one-dimensional and two-dimensional (1)H-NMR. All peptides have a single backbone conformation. The -, psi-angles and chi(1) rotamer populations of tryptophan vary with position in the sequence. The peptides have fluorescence emission maxima of 350-355 nm, quantum yields of 0.04-0.24, and triple exponential fluorescence decays with lifetimes of 4.4-6.6, 1.4-3.2, and 0.2-1.0 ns at 5 degrees C. Lifetimes were correlated with ground-state conformers in six peptides by assigning the major lifetime component to the major NMR-determined chi(1) rotamer. In five peptides the chi(1) = -60 degrees rotamer of tryptophan has lifetimes of 2.7-5.5 ns, depending on local backbone conformation. In one peptide the chi(1) = 180 degrees rotamer has a 0.5-ns lifetime. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence. 相似文献
10.
Through steady-state and time-resolved fluorescence experiments, the polarity of the bilayers of egg phosphatidylcholine vesicles was studied by means of the solvatochromic 2-anthroyl fluorophore which we have recently introduced for investigating the environmental micropolarity of membranes and which was incorporated synthetically in phosphatidylcholine molecules (anthroyl-PC) in the form of 8-(2-anthroyl)octanoic acid. Fluorescence quenching experiments carried out with N,N-dimethylaniline and 12-doxylstearic acid as quenchers showed that the 2-anthroyl chromophore was located in depth in the hydrophobic region of the lipid bilayer corresponding to the C9-C16 segment of the acyl chains. Steady-state fluorescence spectroscopy revealed a nonstructured and red-shifted (lambda em(max) = 464 nm) spectrum for the probe in egg-PC bilayers, which greatly differed from the structured and blue (lambda em(max) = 404 nm) spectrum the fluorophore was shown to display in n-hexane. While the fluorescence decays of the fluorophore in organic solvents were monoexponential, three exponentials were required to account for the fluorescence decays of anthroyl-PC in egg-PC vesicles, with average characteristic times of 1.5 ns, 5.5 ns, and 20 ns. These lifetime values were independent of the emission wavelength used. Addition of cholesterol to the lipid did not alter these tau values. One just observed an increase in the fractional population of the 1.5-ns short-living species detrimental to the population of the 20-ns long-living ones. These observations enabled time-resolved fluorescence spectroscopy measurements to be achieved in the case of the 1/1 (mol/mol) egg-PC/cholesterol mixture. Three distinct decay associated spectra (DAS) were recorded, with maximum emission wavelengths, respectively, of 410 nm, 440 nm, and 477 nm for the 1.5-ns, 6-ns, and 20-ns lifetimes found in this system. On account of the properties and the polarity scale previously established for the 2-anthroyl chromophore in organic solvents, these data strongly suggest the occurrence of three distinct excited states for anthroyl-PC in egg-PC bilayers, corresponding to three environments for the 2-anthroyl chromophore, differing in polarity. The lifetime of 1.5 ns and the corresponding structured and blue (lambda em(max) = 410 nm) DAS account for a hydrophobic environment, with an apparent dielectric constant of 2, which is that expected for the hydrophobic core of the lipid bilayer.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
11.
Time-resolved fluorescence studies have been performed on (+)-anti-7,8-dihydrodiol-9,10-epoxybenzo[a]pyrene adducts in double-stranded poly(dG-dC).(dG-dC). Part of the adduct population gives rise to excimer fluorescence. The heterogeneous fluorescence emission decay curves at 22 degrees C could be resolved into three components with lifetimes: 0.4 ns, 3 ns and 24 ns for the total fluorescence (monomer and excimer emission), and 0.5 ns, 5 ns and 24 ns, respectively, for excimer emission alone. The relative amplitudes for the longer lifetimes were larger for the pure excimer population than for the mixed population. The fluorescence polarization anisotropy decay curves were resolved into two components of rotational correlation times: 0.4 ns and 25 ns for the total fluorescence and 0.3 ns and 33 ns for the excimer fluorescence. We interpret the two rotational correlation times to correspond to local motion of the adduct and segmental motion of the polynucleotide, respectively. 相似文献
12.
Pressure and low temperature effects on the fluorescence emission spectra and lifetimes of the photosynthetic components of cyanobacteria.
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The effects of hydrostatic pressure on the excited state reactions of the photosynthetic system of cyanobacteria were studied with the use of stationary and dynamic fluorescence spectroscopy. When the cells were excited with blue light (442 nm), hydrostatic pressure promoted a large increase in the fluorescence emission of the phycobilisomes (PBS). When PBS were excited at 565 nm, the shoulder originating from photosystem II (PSII) emission (F685) disappeared under 2.4 kbar compression, suggesting suppression of the energy transfer from PBS to PSII. At atmospheric pressure, the excited state decay was complex due to energy transfer processes, and the best fit to the data consisted of a broad Lorentzian distribution of short lifetimes. At 2.4 kbar, the decay data changed to a narrower distribution of longer lifetimes, confirming the pressure-induced suppression of the energy transfer between the PBS and PSII. When the cells were excited with blue light, the decay at atmospheric pressure was even more complex and the best fit to the data consisted of a two-component Lorentzian distribution of short lifetimes. Under compression, the broad distribution of lifetimes spanning the region 100-1,000 ps disappeared and gave rise to the appearance of a narrow distribution characteristic of the PBS centered at 1.2 ns. The emission of photosystem I underwent 2.2-fold increase at 2.4 kbar and room temperature. A decrease in temperature from 20 to -10 degrees C at 2.4 kbar promoted a further increase in the fluorescence emission from photosystem I to a level comparable with that obtained at temperatures below 120 degrees K and atmospheric pressure. On the other hand, when the temperature was decreased under pressure, the PBS emission diminished to very low value at blue or green excitation, suggesting the disassembly into the phycobiliprotein subunits. 相似文献
13.
Hydrophobic surfaces of tubulin probed by time-resolved and steady-state fluorescence of nile red 总被引:3,自引:0,他引:3
Binding of Nile Red to tubulin enhances and blue-shifts fluorescence emission to about 623 nm with a "shoulder" around 665 nm. Binding is reversible and saturable with an apparent Kd of approximately 0.6 microM. Nile Red does not alter tubulin polymerization, and polymerization in 2-(N-morpholino)ethanesulfonic acid (Mes) buffer does not alter the spectrum of the Nile Red-tubulin complex. In contrast, polymerization in glutamate buffer results in a red shift, reduction of intensity, and a decrease in lifetime, suggesting an increase in "polarity" of the binding environment. Lifetimes of 4.5 and 0.6 ns fluorescence in Mes buffer are associated with the 623-nm peak and the 665-nm shoulder, respectively. Indirect excitation spectra for these components are distinct and the 4.5-ns component exhibits tryptophan to Nile Red energy transfer. Acrylamide quenching yields linear Stern-Volmer plots with unchanged lifetimes, indicating static quenching. Apparent quenching constants are wavelength-dependent; global analysis reveals a quenchable component corresponding to the 4.5 ns component and an "unquenchable" component superposing the 0.6-ns spectrum. Analysis of anisotropy decay required an "associative" model which yielded rotational correlation times of greater than 50 ns for the 4.5-ns lifetime and 0.3 ns for the 0.6-ns lifetime. Dilution of tubulin in Mes results in an apparent red shift of emission without lifetime changes, due only to loss of the 623-nm component. These data are reconciled in terms of a model with two binding sites on the tubulin dimer. The more "nonpolar" site is located in a region of subunit-subunit contact which accounts for the fluorescence changes upon dilution; this permits estimation of a subunit dissociation constant of 1 microM. 相似文献
14.
M Consuelo Jiménez Miguel A Miranda Rosa Tormos Ignacio Vayá 《Photochemical & photobiological sciences》2004,3(11-12):1038-1041
The photophysical properties of S-flurbiprofen [S-2-fluoro-alpha-methyl-4-biphenylacetic acid], a nonsteroidal anti-inflammatory drug, have been examined using steady-state and time-resolved spectroscopic techniques. The energy of its first singlet excited state is 99 kcal mol(-1). The fluorescence quantum yields and lifetimes (at 300 nm) have been determined in acetonitrile, methanol, hexane and PBS; they are in the range 0.15相似文献
15.
The binding of cyclosporine to human peripheral blood lymphocytes (PBLs) was studied by measuring the fluorescence emission spectrum and lifetime of the fluorescent and immunosuppressive cyclosporine derivative dansyl-cyclosporine (DCs). The emission maximum and fluorescence lifetime of DCs were characterized in several solvents. The fluorescence emission maximum and lifetime of DCs increased at a high dielectric constant. The fluorescence lifetime decay curve of DCs was a monoexponential function in all solvents tested. Fluorescence micrographs of lipid vesicles and erythrocytes labeled with DCs exhibit uniform staining patterns, whereas PBLs show heterogeneous DCs labeling. DCs exhibits a relatively low emission maximum (490 nm) in erythrocyte membranes. Such an emission maximum is characteristic of a hydrophobic environment. DCs in PBLs also has a low emission maximum (484 nm). The lifetime of DCs in PBLs required two exponential terms to properly fit the lifetime decay curve and could not be attributed to light scattering. One short component (4.7 +/- 1.0 ns) and a second long component (18.5 +/- 1.0 ns) were resolved from the DCs fluorescence decay curves. Time-resolved anisotropy of DCs in PBLs revealed that the labeled drug was present in an anisotropic environment, consistent with at least some DCs being bound to a membrane. These fluorescence studies suggest that DCs interacts with multiple and/or heterogeneous sites in peripheral blood lymphocytes. 相似文献
16.
Studies on Chromophore Coupling in Isolated Phycobiliproteins: II. Picosecond Energy Transfer Kinetics and Time-Resolved Fluorescence Spectra of C-Phycocyanin from Synechococcus 6301 as a Function of the Aggregation State
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The fluorescence kinetics of C-Phycocyanin in the monomeric, trimeric, and hexameric aggregation states has been measured as a function of the emission wavelength with picosecond resolution using the single-photon timing technique. All the decay curves measured at the various emission wavelengths were analyzed simultaneously by a global data analysis procedure. A sum of four exponentials was required to fit the data for the monomers and trimers. Only in the case of the hexamers, a three-exponential model function proved to be nearly sufficient to describe the experimental decays. The lifetime of those fluorescence components reflecting energy transfer decreased with increasing aggregation. This is due to the increased number of efficient acceptor molecules next to a donor in the higher aggregates. In all aggregates the shortest-lived component, ranging from 50 ps for monomer to 10 ps for hexamers, is observed as a decay term (positive amplitude) at short emission wavelength. At long emission wavelength it turns into a rise term (negative amplitude). The lifetime of a second ps-component ranges from 200 ps for monomers to 50 ps for hexamers. The long-lived (ns) fluorescence is inhomogeneous in monomers and trimers, showing two lifetimes of ~0.6 and 1.3 ns. The latter one carries the larger amplitude. The amplitudes of the kinetic components in the fluorescence decays are presented as time-resolved component spectra. A theoretical model has been derived to rationalize the observed fluorescence kinetics. Using symmetry arguments, it is shown that the fluorescence kinetics of C-Phycocyanin is expected to be characterized by three exponential kinetic components, independent of the aggregation state. An analytical expression is derived, which allows us to gain a detailed understanding of the origin of the different kinetic components and their associated time-resolved spectra. Numerical calculations of time-resolved spectra are compared with the experimental data. 相似文献
17.
Decay kinetics and quantum yields of fluorescence in photosystem I from Synechococcus elongatus with P700 in the reduced and oxidized state: are the kinetics of excited state decay trap-limited or transfer-limited?
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Transfer and trapping of excitation energy in photosystem I (PS I) trimers isolated from Synechococcus elongatus have been studied by an approach combining fluorescence induction experiments with picosecond time-resolved fluorescence measurements, both at room temperature (RT) and at low temperature (5 K). Special attention was paid to the influence of the oxidation state of the primary electron donor P700. A fluorescence induction effect has been observed, showing a approximately 12% increase in fluorescence quantum yield upon P700 oxidation at RT, whereas at temperatures below 160 K oxidation of P700 leads to a decrease in fluorescence quantum yield ( approximately 50% at 5 K). The fluorescence quantum yield for open PS I (with P700 reduced) at 5 K is increased by approximately 20-fold and that for closed PS I (with P700 oxidized) is increased by approximately 10-fold, as compared to RT. Picosecond fluorescence decay kinetics at RT reveal a difference in lifetime of the main decay component: 34 +/- 1 ps for open PS I and 37 +/- 1 ps for closed PS I. At 5 K the fluorescence yield is mainly associated with long-lived components (lifetimes of 401 ps and 1.5 ns in closed PS I and of 377 ps, 1.3 ns, and 4.1 ns in samples containing approximately 50% open and 50% closed PS I). The spectra associated with energy transfer and the steady-state emission spectra suggest that the excitation energy is not completely thermally equilibrated over the core-antenna-RC complex before being trapped. Structure-based modeling indicates that the so-called red antenna pigments (A708 and A720, i.e., those with absorption maxima at 708 nm and 720 nm, respectively) play a decisive role in the observed fluorescence kinetics. The A720 are preferentially located at the periphery of the PS I core-antenna-RC complex; the A708 must essentially connect the A720 to the reaction center. The excited-state decay kinetics turn out to be neither purely trap limited nor purely transfer (to the trap) limited, but seem to be rather balanced. 相似文献
18.
Tryptophan fluorescence of terminal deoxynucleotidyl transferase: effects of quenchers on time-resolved emission spectra 总被引:2,自引:0,他引:2
Terminal deoxynucleotidyl transferase (EC 2.7.7.31) is a eucaryotic DNA polymerase that does not require a template. The tryptophan environments in calf thymus terminal transferase were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was separated by time-resolved emission spectroscopy. Nanosecond fluorescence decays at 296-nm excitation and various emission wavelengths were deconvolved by global analysis, assuming that the lifetimes but not the relative weighting factors were independent of emission wavelength. The data were fit to three exponentials of lifetimes tau 1 = 1.4 ns, tau 2 = 4.5 ns, and tau 3 = 7.7 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 328, 335, and 345 nm. The accessibility of individual tryptophan environments to polar and nonpolar fluorescence quenchers was examined in steady-state and time-resolved experiments. In the presence of iodide and acrylamide, the steady-state emission spectra shift to the blue. However, at low quencher concentrations, the emission from the 7.7-ns component (maximum 345 nm) is hardly affected, suggesting that this hydrophilic tryptophan environment is buried within the protein. On the other hand, the red shift in the steady-state emission spectrum in the presence of trichloroethanol indicates that the 1.4-ns component (maximum 328 nm) is an exposed hydrophobic tryptophan environment. The results are consistent with an inside-out model for terminal transferase protein, with the more hydrophobic tryptophan(s) near the surface and the most hydrophilic tryptophan(s) in the core. 相似文献
19.
One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins
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We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant approximately 3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were approximately 1.1 ns (main component) and approximately 3.3 ns. The anisotropy decay of WT-GFP and S65T-GFP was also monoexponential (global rotational correlation time of 16 +/- 1 ns). The approximately 1.1 ns lifetime of RSGFP was associated with a faster rotational depolarization, evaluated as an additional approximately 13 ns component. This feature we attribute tentatively to a greater rotational freedom of the anionic chromophore. With OPE, the initial anisotropy was close to the theoretical limit of 0.4; with TPE it was higher, approaching the TPE theoretical limit of 0.57 for the colinear case. The measured power dependence of the fluorescence signals provided direct evidence for TPE. The general independence of fluorescence decay times, rotation correlation times, and steady-state emission spectra on the excitation mode indicates that the fluorescence originated from the same distinct excited singlet states (A*, I*, B*). However, we observed a relative enhancement of blue fluorescence peaked at approximately 440 nm for TPE compared to OPE, indicating different relative excitation efficiencies. We infer that the two lifetimes of RSGFP represent the deactivation of two substates of the deprotonated intermediate (I*), distinguished by their origin (i.e., from A* or B*) and by nonradiative decay rates reflecting different internal environments of the excited-state chromophore. 相似文献
20.
Energy transfer and distribution in the red alga Porphyra perforata studied using picosecond fluorescence spectroscopy 总被引:1,自引:0,他引:1
The detailed process of excitation transfer among the antenna pigments of the red alga Porphyra perforata was investigated by measuring time-resolved fluorescence emission spectra using a single-photon timing system with picosecond resolution. The fluorescence decay kinetics of intact thalli at room temperature revealed wavelength-dependent multi-component chlorophyll a fluorescence emission. Our analysis attributes the majority of chlorophyll a fluorescence to excitation originating in the antennae of PS II reaction centers and emitted with maximum intensities at 680 and 740 nm. Each of these fluorescence bands was characterized by two kinetic decay components, with lifetimes of 340-380 and 1700-2000 ps and amplitudes varying with wavelength and the photochemical state of the PS II reaction centers. In addition, a small contribution to the long-wavelength fluorescence band is proposed to arise from chlorophyll a antennae coupled to PS I. This component displays fast decay kinetics with a lifetime of approx. 150 ps. Desiccation of the thalli dramatically increases the contribution of this fast decay component. 相似文献