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1.
Picosecond events in the photochemical cycle of the light-driven chloride-pump halorhodopsin. 总被引:4,自引:0,他引:4
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H J Polland M A Franz W Zinth W Kaiser P Hegemann D Oesterhelt 《Biophysical journal》1985,47(1):55-59
The early events in halorhodopsin after light excitation are studied with picosecond time resolution. Absorption and fluorescence measurements show that the electronically excited state of the incorporated retinal has a lifetime of 5 ps. Within that time a red-shifted photoproduct is formed that remains stable for at least 2 ns. 相似文献
2.
《FEBS letters》1986,198(2):268-272
The kinetics of the fluorescence emitted by the ‘special pair’ of bacteriochlorophyll b molecules in reaction centres from Rhodopseudomonas viridis was recorded in the near infrared, with a time resolution of 1 ns. In nonreduced reaction centres two decay components were resolved with lifetimes of <0.5 and 2.5 ns. Upon reduction of the menaquinone electron acceptor three decay components were detected with lifetimes of < 0.5, 2.5 and 15ns. 相似文献
3.
We describe a new method for the analysis of phase-sensitive fluorescence emission spectra. This method permits the resolution of three-component mixtures using spectra measured at a single modulation frequency. Phase-sensitive spectra are recorded using one modulation frequency, at a number of arbitrary detector phase angles. It is not necessary to suppress any one component. The spectra are then used to estimate the component lifetimes and steady-state fractional intensities using a nonlinear least-squares analysis procedure. The only requirement for the analysis is the knowledge of the steady-state spectra of the individual components. This procedure allowed the resolution of a two-component mixture of 9-methylanthracene (4.5 ns) and 9,10-diphenylanthracene (5.9 ns). It should be noted that resolution of two lifetimes which differ by only 30% is a difficult task. Additionally, we resolved a three-component mixture with lifetimes that differed fourfold: p-bis[2-(5-phenyloxazolyl)]benzene (1.3 ns), 9-methylanthracene (4.5 ns), and 9,10-diphenylanthracene (5.9 ns). Conveniently, the technique utilizes a commercially available fixed-frequency phase fluorometer. 相似文献
4.
Sandra Mitic Jan W. van Nieuwkasteele Albert van den Berg Simon de Vries 《Analytical biochemistry》2015
Unravelling (bio)chemical reaction mechanisms and macromolecular folding pathways on the (sub)microsecond time scale is limited by the time resolution of kinetic instruments for mixing reactants and observation of the progress of the reaction. To improve the mixing time resolution, turbulent four- and two-jet tangential micro-mixers were designed and characterized for their mixing and (unwanted) premixing performances employing acid–base reactions monitored by a pH-sensitive fluorescent dye. The mixing performances of the micro-mixers were determined after the mixing chamber in a free-flowing jet. The premixing behavior in the vortex chamber was assessed in an optically transparent glass–silicon replica of a previously well-characterized stainless-steel four-jet tangential micro-mixer. At the highest flow rates, complete mixing was achieved in 160 ns with only approximately 9% premixing of the reactants. The mixing time of 160 ns is at least 50 times shorter than estimated for other fast mixing devices. Key aspects to the design of ultrafast turbulent micro-mixers are discussed. The integration of these micro-mixers with an optical flow cell would enable the study of the very onset of chemical reactions in general and of enzyme catalytic reactions in particular. 相似文献
5.
The time course of ligand recombination to the myoglobin from Aplysia limacina, which has Val(E7), was measured following photolysis by flashes of 35 ps to 300 ns with a time resolution of 10 ps or 1 ns. CO shows only biomolecular recombination. O2 has a small geminate reaction with a half-time of tens of picoseconds, but no nanosecond geminate reaction. NO has two picosecond relaxations with half-times of 70 ps (15%) and 1 ns (80%) and one nanosecond relaxation with a half-time of 4.6 ns. The biomolecular rates for O2 and NO are the same: 2 x 10(7) M-1 s-1. Methyl and ethyl isonitriles have a geminate reaction with a half-time of 35 ps. Ethyl isonitrile has, in addition, a nanosecond relaxation (25%) with a half-time of 100 ns. t-Butyl isonitrile has four geminate relaxations (10 ps, 35 ps, 1 ns, and 1 microseconds). Analysis of the results suggests much easier movement of ligand between the heme pocket and the exterior than in sperm whale myoglobin (His(E7]. The reactivity of the heme is little different, placing the effect of the differences from sperm whale myoglobin on the distal side of the heme. 相似文献
6.
Rappaport Fabrice Béal Daniel Verméglio André Joliot Pierre 《Photosynthesis research》1998,55(2-3):317-323
We have studied the electron transfer reactions from the tetraheme cytochrome of Rhodopseudomonas viridis to the oxidized primary donor in whole cells with a new high sensitivity spectrophotometer. In this apparatus the monochromatic detecting flashes are provided by a YAG pumped Optical Parametric Oscillator, allowing a 10 ns time resolution. When four hemes are reduced the observed electron transfer reaction sequence is the following: first the low-potential c552 heme (the number refers to the maximum absorption wavelength in the alpha-band region) is oxidized with a half time of 130 ns, in agreement with previous reports of measurements performed with purified reaction centers. Then, the electron hole is transferred to the low potential c554 heme with a half time of 2.6 µs. When only the two high potential hemes are reduced the observed electron transfer sequence is the following: oxidation of the high potential c559 heme in the hundreds of ns time range (410 ns), reduction of this heme by the high potential c556 heme in the µs time range (2.7 µs). This confirms the first steps of electron transfer observed in isolated reaction centers. However, in the microsecond time domain, the overall amount of oxidized hemes increases suggesting that, in vivo, the equilibrium constant between the P+/P and the c559ox/c559red couples is significantly lower than expected from the difference in their midpoint potentials. 相似文献
7.
Resonance Raman kinetic spectroscopy of bacteriorhodopsin on the microsecond time scale. 总被引:1,自引:0,他引:1
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Using a rotating disk with a slit of variable width, a continuous wave argon ion laser, and an Optical Multichannel Analyzer for detection, a new technique is reported which should, in principle, be capable of recording resonance Raman spectra with time resolution of 100 ns. The resonance Raman spectra of the intermediates of the photosynthetic cycle of bacteriorhodopsin are recorded on the microsecond time scale. Both the kinetic results and the resonance enhancement profile suggest that deprotonation results in an intermediate preceding bM412 that has an optical absorption maximum at a wavelength longer than that of bM412. 相似文献
8.
Kinetics of phyllosemiquinone oxidation in the Photosystem I reaction centre of Acaryochloris marina
Santabarbara S Bailleul B Redding K Barber J Rappaport F Telfer A 《Biochimica et biophysica acta》2012,1817(2):328-335
Light-induced electron transfer reactions in the chlorophyll a/d-binding Photosystem I reaction centre of Acaryochloris marina were investigated in whole cells by pump-probe optical spectroscopy with a temporal resolution of ~5ns at room temperature. It is shown that phyllosemiquinone, the secondary electron transfer acceptor anion, is oxidised with bi-phasic kinetics characterised by lifetimes of 88±6ns and 345±10ns. These lifetimes, particularly the former, are significantly slower than those reported for chlorophyll a-binding Photosystem I, which typically range in the 5-30ns and 200-300ns intervals. The possible mechanism of electron transfer reactions in the chlorophyll a/d-binding Photosystem I and the slower oxidation kinetics of the secondary acceptors are discussed. 相似文献
9.
《BBA》1987,893(2):320-332
The primary charge separation in Photosystem I of pea chloroplasts was measured as a photovoltage in the pico- and nanosecond time range by applying laser flashes at 532 nm of variable energy and different duration (12 ns and 30 ps, respectively). Contributions to the photovoltage from Photosystem II was eliminated by addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination. The dependence of the photovoltage amplitude on the excitation energy could be described by an exponential saturation law when the excitation flash had a duration of 12 ns. Nearly the same dependence was found when the excitation source was the train of a mode-locked laser (approx. ten 30-ps flashes spaced by 7 ns; highest energy of a single flash, 80 μJ / cm−2). Even with single 30-ps flashes the photovoltage was only slightly smaller than the one elicited by 12-ns flashes of the same energy. These findings demonstrate that trapping of excitation energy by the reaction center of Photosystem I is much more effective than losses by annihilation and other loss processes. The photovoltage yield was nearly independent of the fraction of closed traps, thus demonstrating that the absorption cross section of Photosystem I is not altered by the closing of its reaction centers. By recording the rise time of the photovoltage with our highest time resolution we found that the trapping rate of the excitation energy in Photosystem I depended on the energy of the 30-ps flashes: at low excitation energies (less than 1014 photons / cm2 per pulse) trapping occurred within 90 ± 15 ps and at high excitation energy (1015 photons / cm2 per pulse) trapping and charge stabilization occurred within the time resolution of the apparatus, i.e., up to 50 ps. The trapping rate at low energies is in agreement with the one determined by fluorescence decay kinetics. Up to 50 ns there was no further detectable electrogenic phase (neither forward nor backward reactions). This demonstrates that all the electrogenicity, produced by the charge separation, takes place in less than 50 ps. 相似文献
10.
Z Ren B Perman V Srajer T Y Teng C Pradervand D Bourgeois F Schotte T Ursby R Kort M Wulff K Moffat 《Biochemistry》2001,40(46):13788-13801
The photocycle of the bacterial blue-light photoreceptor, photoactive yellow protein, was stimulated by illumination of single crystals by a 7 ns laser pulse. The molecular events were recorded at high resolution by time-resolved X-ray Laue diffraction as they evolved in real time, from 1 ns to seconds after the laser pulse. The complex structural changes during the photocycle at ambient temperature are displayed in a movie of difference electron density maps relative to the dark state. The step critical to entry into the photocycle is identified as flipping of the carbonyl group of the 4-hydroxycinnamic acid chromophore into an adjacent, hydrophobic environment rather than the concomitant isomerization about the double bond of the chromophore tail. The structural perturbation generated at the chromophore propagates throughout the entire protein as a light-induced "protein quake" with its "epicenter" at the carbonyl moiety of the chromophore. 相似文献
11.
Gauden M Yeremenko S Laan W van Stokkum IH Ihalainen JA van Grondelle R Hellingwerf KJ Kennis JT 《Biochemistry》2005,44(10):3653-3662
The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal domain belonging to a new class of photoreceptors designated BLUF domains. AppA was shown to control photosynthesis gene expression in response to blue light and oxygen tension. We have investigated the photocycle of the AppA BLUF domain by ultrafast fluorescence, femtosecond transient absorption, and nanosecond flash-photolysis spectroscopy. Time-resolved fluorescence experiments revealed four components of flavin adenine dinucleotide (FAD) excited-state decay, with lifetimes of 25 ps, 150 ps, 670 ps, and 3.8 ns. Ultrafast transient absorption spectroscopy revealed rapid internal conversion and vibrational cooling processes on excited FAD with time constants of 250 fs and 1.2 ps, and a multiexponential decay with effective time constants of 90 ps, 590 ps, and 2.7 ns. Concomitant with the decay of excited FAD, the rise of a species with a narrow absorption difference band near 495 nm was detected which spectrally resembles the long-living signaling state of AppA. Consistent with these results, the nanosecond flash-photolysis measurements indicated that formation of the signaling state was complete within the time resolution of 10 ns. No further changes were detected up to 15 micros. The quantum yield of the signaling-state formation was determined to be 24%. Thus, the signaling state of the AppA BLUF domain is formed on the ultrafast time scale directly from the FAD singlet excited state, without any apparent intermediate, and remains stable over 12 decades of time. In parallel with the signaling state, the FAD triplet state is formed from the FAD singlet excited state at 9% efficiency as a side reaction of the AppA photocycle. 相似文献
12.
Plasma membrane voltage changes during nanosecond pulsed electric field exposure 总被引:1,自引:0,他引:1
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Frey W White JA Price RO Blackmore PF Joshi RP Nuccitelli R Beebe SJ Schoenbach KH Kolb JF 《Biophysical journal》2006,90(10):3608-3615
The change in the membrane potential of Jurkat cells in response to nanosecond pulsed electric fields was studied for pulses with a duration of 60 ns and maximum field strengths of approximately 100 kV/cm (100 V/cell diameter). Membranes of Jurkat cells were stained with a fast voltage-sensitive dye, ANNINE-6, which has a subnanosecond voltage response time. A temporal resolution of 5 ns was achieved by the excitation of this dye with a tunable laser pulse. The laser pulse was synchronized with the applied electric field to record images at times before, during, and after exposure. When exposing the Jurkat cells to a pulse, the voltage across the membrane at the anodic pole of the cell reached values of 1.6 V after 15 ns, almost twice the voltage level generally required for electroporation. Voltages across the membrane on the side facing the cathode reached values of only 0.6 V in the same time period, indicating a strong asymmetry in conduction mechanisms in the membranes of the two opposite cell hemispheres. This small voltage drop of 0.6-1.6 V across the plasma membrane demonstrates that nearly the entire imposed electric field of 10 V/mum penetrates into the interior of the cell and every organelle. 相似文献
13.
We describe the construction and performance of a variable-frequency phase-modulation fluorometer. This instrument, which provides modulation frequencies from 1 to 200 MHz, was constructed using commercially available components. To facilitate the introduction of these instruments into other laboratories we describe in detail the chosen components and the principles of operation. The present light source is a continuous-wave helium-cadmium laser, which provides convenient excitation wavelengths of 325 and 442 nm. Modulation of the incident light is provided by one of several electro-optic modulators. The extent of modulation ranges from 1.0 to 0.2 as the frequency increases from 1 to 200 MHz. Phase angles and demodulation factors are measured using the cross-correlation method. The closely spaced frequencies are provided by two direct frequency synthesizers. The phase and modulation measurements are accurate to 0.2 degrees and 0.002, respectively, from 1 to 200 MHz. This accuracy allows considerable resolution of complex decay laws. The usefulness of frequency-domain fluorometry for the resolution of multiexponential decays is illustrated by the analysis of several difficult mixtures. As examples, we resolved a two-component mixture of anthracene (4.1 ns) and 9,10-diphenylanthracene (6.3 ns), and confirmed that the intensity decay of NADH in aqueous buffer is at least a double exponential (0.2 and 0.86 ns). We also resolved an especially difficult mixture of anthracene (4.1 ns) and 9-methylanthracene (4.5 ns), and a three-component mixture with decay times of 1.3, 4.1 and 7.7 ns. Frequency-domain fluorometers appear to be particularly useful for determination of complex decays of fluorescence anisotropy. This capability is illustrated by the determination of rotational correlation times as short as 47 ps for p-bis[2-(5-phenyloxazolyl)]benzene (POPOP) in hexane at 40 degrees C, and by the resolution of the two correlation times of anisotropic rotators such as perylene and 9-aminoacridine. Resolution of two anisotropy decay times for 9-aminoacridine is a difficult test because these correlation times differ by less than 2-fold. The resolution of multiexponential decays of intensity and anisotropy possible with this instrument is at least equivalent to that obtained using state-of-the-art time-resolved instruments based on mode-locked laser sources. The ease and rapidity of frequency-domain measurements, the relative simplicity of the equipment, the accuracy of the measurements and the lack of significant systematic errors indicate that frequency-domain fluorometry will be widely useful in chemical and biochemical research. 相似文献
14.
Enzyme dynamics and activity: time-scale dependence of dynamical transitions in glutamate dehydrogenase solution.
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We have examined the temperature dependence of motions in a cryosolution of the enzyme glutamate dehydrogenase (GDH) and compared these with activity. Dynamic neutron scattering was performed with two instruments of different energy resolution, permitting the separate determination of the average dynamical mean square displacements on the sub-approximately 100 ps and sub-approximately 5 ns time scales. The results demonstrate a marked dependence on the time scale of the temperature profile of the mean square displacement. The lowest temperature at which anharmonic motion is observed is heavily dependent on the time window of the instrument used to observe the dynamics. Several dynamical transitions (inflexions of the mean squared displacement) are observed in the slower dynamics. Comparison with the temperature profile of the activity of the enzyme in the same solvent reveals dynamical transitions that have no effect on GDH function. 相似文献
15.
V. F. Tarasenko E. Kh. Baksht A. G. Burachenko I. D. Kostyrya M. I. Lomaev V. K. Petin D. V. Rybka S. V. Shlyakhtun 《Plasma Physics Reports》2008,34(12):1028-1036
The spectra of an ultrashort avalanche electron beam generated by a nanosecond discharge in atmospheric-pressure air were investigated. The temporal characteristics of the beam current pulses, gap voltage, and discharge current in a gas diode were measured with a time resolution of ~0.1 ns. A simple technique was developed for recovering electron spectra from the curves of beam attenuation by aluminum foils. The effect of the cathode design, electrode gap length, and generator parameters on the electron spectra were studied using seven setups. It is shown that generation of electrons with anomalously high energies requires the use of cathodes with increased curvature radius. 相似文献
16.
Kinetics of charge transfer at the lipid bilayer-water interface on the nanosecond time scale. 总被引:2,自引:0,他引:2
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Advances in instrumentation allow electrical measurements across the planar lipid bilayer to be made with nanosecond time resolution. The electron transfer reaction between photoexcited magnesium octaethylporphyrin in the lipid to a variety of ionically charged acceptors in the water is found to be purely dynamic over a wide range of concentrations of acceptors and up to the time constant of the apparatus, 4 ns. The saturation of the amplitude of the photovoltage with increasing concentration of acceptor is caused by the finite lifetime of the excited state, not by formation of a static pigment-acceptor complex. The reactions are an excellent probe of the lipid-water interface over an extended time scale. No appreciable barrier to reaction exists at this interface beyond the 5-ns time. That is, any water or choline group structure may be evanescent on this time scale. Electrostatic interactions indicate that the acceptor molecules penetrate to the level of the phosphocholine groups with differing orientations. It will be possible to extend the time scale into the picosecond range by decreasing the response time and by deconvolutions. 相似文献
17.
Needles from spruces at different environmental and physiological conditions were analyzed by picosecond fluorescence spectroscopy using a novel laser diode and single photon counting detection. The decay curves of chlorophyll fluorescence showed a superposition of three exponentially decaying components with time constants of T1 = 100-200 ps, T2 = 300-500 ps and T3 = 2.0-3.5 ns. A high relative intensity of the long-lived component was found in damaged spruces as well as in trees showing first symptoms of yellowing, needle loss or parasite infection, although all measurements were carried out with green needles which appeared visually intact. Therefore, fluorescence spectroscopy with subnanosecond time resolution seems to be a valuable attempt for an early detection of forest decline. 相似文献
18.
We used frequency-domain fluorescence spectroscopy to measure the fluorescence lifetime and anisotropy decays of indole in propylene glycol, and of the tryptophan emission of melittin monomer and tetramer in water solutions at 5 degrees C. We obtained an increase in resolution of the anisotropy decays by using multiple excitation wavelengths, chosen to provide a range of fundamental anisotropy values. The multi-excitation wavelength anisotropy decays were analyzed globally to recover a single set of correlation times with wavelength-dependent anisotropy amplitudes. Simulated data and kappaR2 surfaces are shown to reveal the effect of multi-wavelength data on the resolution of complex anisotropy decays. For both indole and melittin, the anisotropy decays are heterogeneous and require two correlation times to fit the frequency-domain data. For indole in propylene glycol at 5 degrees C we recovered correlation times of 0.59 and 4.10 ns, which appear to be characteristic of the rigid and asymmetric indole molecule. For melittin monomer the correlation times were 0.13 and 1.75 ns, and for melittin tetramer 0.12 and 3.96 ns. The shorter and longer correlation times of melittin are due to segmental motions and overall rotational diffusion of the polypeptide. 相似文献
19.
Chlorophyll a fluorescence as a monitor of nanosecond reduction of the photooxidized primary donor P-680 Of photosystem II 总被引:2,自引:0,他引:2
1. Changes in the fluorescence yield of aerobic Chlorella vulgaris have been measured in laser flashes of 15 ns, 30 ns and 350 ns half time. The kinetics after the first flash given after a 3 min dark period could be simulated on a computer using the hypothesis that the oxidized acceptor Q and primary donor P+ are fluorescence quenchers, and Q- is a weak quencher, and that the reduction time for P+ is 20-35 ns. 2. The P+ reduction time for at least an appreciable part of the reaction centers was found to be longer after the second and subsequent flashes. In the first 5 flashes an oscillation was observed. Under steady state conditions, with a pulse separation of 3 s, a reduction time for P+ of about 400 ns for all reaction centers gave the best correspondence between computed and experimental fluorescence kinetics. 相似文献
20.
Michael W. Clarkson Ming Lei Elan Z. Eisenmesser Wladimir Labeikovsky Alfred Redfield Dorothee Kern 《Journal of biomolecular NMR》2009,45(1-2):217-225
Protein motions on all timescales faster than molecular tumbling are encoded in the spectral density. The dissection of complex protein dynamics is typically performed using relaxation rates determined at high and ultra-high field. Here we expand this range of the spectral density to low fields through field cycling using the nucleocapsid protein of the SARS coronavirus as a model system. The field-cycling approach enables site-specific measurements of R 1 at low fields with the sensitivity and resolution of a high-field magnet. These data, together with high-field relaxation and heteronuclear NOE, provide evidence for correlated rigid-body motions of the entire β-hairpin, and corresponding motions of adjacent loops with a time constant of 0.8 ns (mesodynamics). MD simulations substantiate these findings and provide direct verification of the time scale and collective nature of these motions. 相似文献