A subnanosecond resolving spectrofluorimeter for the analysis of protein fluorescence kinetics

A spectrofluorometer is described consisting of an excitation source, optics, detector and time resolving electronics. The excitation source consists of a mode-locked Ar ion laser, synchronously pumps a dye laser, followed by a frequency doubling device. The repetition frequency of the U.V. pulses (FWHM some ps) has been reduced by an extra-cavity electro-optical modulator. Provisions have been made in the optical configuration to determine both time-resolved fluorescence spectra and fluorescence anisotropy decay curves. The commercially avialable electronics have been optimized for maximum time resolution. The spectral output of the excitation source is confined between 280 and 310 nm, which encompasses the region for eliciting protein fluorescence. The performance of the complete system has been tested with single lifetime standards line p-terphenyl in cyclohexane or with $\text{N-}\text{acetyl}\text{-}\text{L}\text{-}\text{tryptophanamide}$ in pH 7.5 buffer. Serum albumins from human and bovine sources have been employed as examples for time resolved fluorescence spectra and for the demonstration of anisotropy decay curves. Using these methods protein dynamics in the (sub)nanosecond time region can be directly explored.     

Application of a reference convolution method to tryptophan fluorescence in proteins. A refined description of rotational dynamics

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.     

Rapid relaxation processes in pig heart lipoamide dehydrogenase revealed by subnanosecond resolved fluorometry

The decay kinetics of the FAD-fluorescence in lipoamide dehydrogenase from pig heart have been reinvestigated using phase fluorometric methods and sophisticated laser pulse techniques. Both pulse and modulation methods lead to distinct heterogeneity in lifetimes. The two different techniques lead to good correspondence in the longer lifetime component of a biexponential decay model, whereas the more rapidly decaying component is distinctly shorter and has a larger amplitude using the phase technique with two available modulation frequencies (15 and 60 MHz). Lifetime measurements as a function of temperature and in the presence of D₂O instead of H₂O illustrate that the quenching of the FAD fluorescence in lipoamide. dehydrogenase is mainly dynamic in nature and that solvent comes into contact with the fluorophor. Mobility of the flavin itself, free and bound to the enzyme, has been measured by both differential polarized phase fluorometry and experimental fluorescence anisotropy decay after ps laser pulse excitation. By employing flavin models it has been shown that both techniques have ps time resolution. Measurements with the latter more direct method indicate a rapid subnanosecond motion of the FAD bound within the enzyme, only visible at temperatures lower than about 15°C, where the protein rotational diffusion is slowed down. The significance of rapid transient conformational fluctuations for catalysis is discussed with reference to recently developed insights reported in the literature.     

Time-resolved fluorescence of apoferritin and its subunits

The decay of the intrinsic fluorescence of the apoferritin polymer and its subunits has been studied by pulse and phase shift techniques. Both techniques show that the fluorescence decay of all the samples tested cannot be described by a single exponential function. The fluorescence decay data of the apoferritin subunits obtained with either technique can be fitted satisfactorily with a function resulting from the sum of two exponential components. However, the polymer data obtained with the high resolution phase shift technique operated either by synchrotron radiation or by a mode-locked argon ion laser can be fitted better using a bimodal gaussian continuous distribution of lifetime components. The molecular basis for this distribution of lifetime values may lie in the heterogeneity of the tryptophan environment generated by the assembly of the subunits into the polymer. The binding of the first 100 irons to apoferritin quenches the intrinsic fluorescence without affecting the lifetimes in a proportional way. This finding may be taken as an indication that the quenching of the tryptophan fluorescence induced by the binding of iron has both static and dynamic components.     

Determination of rotational correlation times from deconvoluted fluorescence anisotropy decay curves. Demonstration with 6,7-dimethyl-8-ribityllumazine and lumazine protein from Photobacterium leiognathi as fluorescent indicators

The experimental and analytical protocols required for obtaining rotational correlation times of biological macromolecules from fluorescence anisotropy decay measurements are described. As an example, the lumazine protein from Photobacterium leiognathi was used. This stable protein (Mr 21 200) contains the noncovalently bound, natural fluorescent marker 6,7-dimethyl-8-ribityllumazine, which has in the bound state a long fluorescence lifetime (tau = 14 ns). Shortening of the fluorescence lifetime to 2.6 ns at room temperature was achieved by addition of the collisional fluorescence quencher potassium iodide. The shortening of tau had virtually no effect on the rotational correlation time of the lumazine protein (phi = 9.4 ns, 19 degrees C). The ability to measure biexponential anisotropy decay was tested by the addition of Photobacterium luciferase (Mr 80 000), which forms an equilibrium complex with lumazine protein. Under the experimental conditions used (2 degrees C) the biexponential anisotropy decay can best be described with correlation times of 20 and 60 ns, representing the uncomplexed and luciferase-associated lumazine proteins, respectively. The unbound 6,7-dimethyl-8-ribityllumazine itself (tau = 9 ns) was used as a model compound for determining correlation times in the picosecond time range. In the latter case rigorous deconvolution from the excitation profile was required to recover the correlation time, which was shorter (100-200 ps) than the measured laser excitation pulse width (500 ps).     

Construction and evaluation of a frequency-domain epifluorescence microscope for lifetime and anisotropy decay measurements in subcellular domains

The measurement of time-resolved fluorescence parameters in living cells provides a powerful approach to study cell structure and dynamics. An epifluorescence microscope was constructed to resolve multi-component fluorescence lifetimes and complex anisotropy decay rapidly in labile biological samples. The excitation source consisted of focused, polarized laser light modulated by an impulse-driven Pockels' cell; parallel acquisition of phase angles and modulation amplitudes at more than 40 frequencies (5-250 MHz) was obtained by multi-harmonic cross-correlation detection. Lifetime decay was measured against standard solutions introduced into the light path proximal to the microscope objective. Anisotropy decay was measured by rotation of a Glan-Thompson polarizer in the emission path. Phase reference light was split from the beam proximal to the microscope. Optical components were selected to avoid depolarization and to optimize fluorescence detection efficiency. The dichoric was replaced by a 1 mm square mirror. Fitting routine statistics were optimized for model discrimination in realistic biological samples. Instrument performance was evaluated using fluorescein in H2O/glycerol and H2O/ethylene glycol mixtures and in Swiss 3T3 fibroblasts in monolayer culture. Objective depolarization effects were evaluated by measurement of anisotropy decay using objectives of different numerical aperture. Lifetime and anisotropy decay measured by microscopy (0.5 micron laser spot) agreed with data obtained by cuvette fluorimetry. New biological applications for time-resolved fluorescence microscopy are discussed.     

Protein rotational motion in solution measured by polarized fluorescence depletion.

A microscope-based system is described for directly measuring protein rotational motion in viscous environments such as cell membranes by polarized fluorescence depletion (PFD). Proteins labeled with fluorophores having a high quantum yield for triplet formation, such as eosin isothiocyanate (EITC), are examined anaerobically in a fluorescence microscope. An acousto-optic modulator generates a several-microsecond pulse of linearly polarized light which produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. When the sample is then probed with light polarized parallel to the excitation pulse, fluorescence recovers over 0-1,000 microseconds as the sum of two exponentials. One exponential corresponds to triplet decay and the other to the rotational relaxation. An exciting pulse perpendicular to the probe beam is then applied. Fluorescence recovery following this pulse is the difference of the same two exponentials. Equations for fluorescence recovery kinetics to be expected in various experimentally significant cases are derived. Least-squares analysis using these equations then permits the triplet lifetime and rotational correlation time to be determined directly from PFD data. Instrumentation for PFD measurements is discussed that permits photobleaching recovery measurements of lateral diffusion coefficients using the same microscope system. With this apparatus, both rotational and translational diffusion coefficients (Dr, Dt) were measured for EITC-labeled bovine serum albumin in glycerol solutions. Values obtained for Dr and Dt are discussed in light of both the PFD models and the experimental system.     

Analysis of fluorescence anisotropy decays by a least square method

A method of fluorescence anisotropy decay analysis is described in this work. The transient anisotropy r(ex)(t) measured in a photocounting pulsefluorimeter is fitted by a non linear least square procedure to the ratio of convolutions of the apparatus response function g(t) by sums of appropriate exponential functions. This method takes rigorously into account the apparatus response function and is applicable to any shape of the later as well as to any values of fluorescence decay times and correlation times. The performances of the method have been tested with data simulated from measured response functions corresponding to an air lamp and a high pressure nitrogen lamp. The statistical standard errors of the anisotropy deca parameters have been found to be smaller than the standard errors previously calculated for the moment method. A systematic error delta in the fluorescence decay time entailed an error deltatheta in the correlation time such as Deltatheta/theta < deltatau/tau. By this method, good fitting of experimental data have been achieved very conveniently and accurately.     

Analysis of time-resolved fluorescence anisotropy in lipid-protein systems

The subnanosecond fluorescence and motional dynamics of the tryptophan residue in the bacteriophage M13 coat protein incorporated within pure dioleoylphosphatidylcholine (DOPC) as well as dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) and dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bilayers (80/20 w/w) with various L/P ratio have been investigated. The fluorescence decay is decomposed into four components with lifetimes of about 0.5, 2.0, 4.5 and 10.0 ns, respectively. In pure DOPC and DOPC/DOPG lipid bilayers, above the phase transition temperature, the rotational diffusion of the protein molecules contributes to the depolarization and the anisotropy of tryptophan is fitted to a dual exponential function. The longer correlation time, describing the rotational diffusion of the whole protein, shortens with increasing temperature and decreasing protein aggregation number. In DMPC/DMPG lipid bilayers, below the phase transition, the rotational diffusion of the protein is slowed down such that the subnanosecond anisotropy decay of tryptophan in this system reflects only the segmental motion of the tryptophan residue. Because of a heterogeneous microenvironment, the anisotropy decay must be described by three exponentials with a constant term, containing a negative coefficient and a negative decay time constant. From such a decay, the tryptophan residue within the aggregate undergoes a more restricted motion than the one exposed to the lipids. At 20 degrees C, the order parameter of the transition moment of the isolated tryptophan is about 0.9 and that for the exposed one is about 0.5.     

Simulation of convoluted and exact emission anisotropy decay profiles

We have Simulated the convolution of the emission anisotropy decay function with both a delta-pulse excitation function (exact solution) and a pulse function of either Gaussian or other functional form. It can be readily shown that convolution with a pulse of finite width leads to lower r0 values (anisotropy at time zero). Especially in the case of short-lived fluorescence, it can be demonstrated that the convoluted anisotropy lags behind the exact anisotropy leading to longer apparent rotational correlation times. Contour plots of r corrections as a function of both fluorescence lifetime and rotational correlation time were constructed for two different pulse profiles. Inspection of these contour diagrams can lead to an estimate of the relative error involved, when anisotropy data are not deconvoluted.     

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.     

Correlation of enzyme activities with fluorescence anisotropy of dansyl-labeled cytochrome b5/NADH-cytochrome-b5 reductase systems in phosphatidylcholine vesicles

The changes in steady-state fluorescence lifetimes and anisotropy decay parameters, as well as enzyme activities, of dansyl-labeled cytochrome b5 (DNS-cytochrome b5), on interaction with NADH-cytochrome-b5 reductase in DMPC vesicles, have been measured as a function of temperature. Steady-state fluorescence of DNS-cytochrome b5 in DMPC vesicles with and without cholesterol was increased on interaction with reductase at temperatures both above and below the DMPC phase transition. In all systems three fluorescence decay components of the dansyl label in DNS-cytochrome b5 were observed. In the reductase-containing system, the long (major) decay time component of DNS-cytochrome b5 and the fraction of the total fluorescence associated with this component increased over the temperature range 15-30 degrees C. In time-resolved anisotropy measurements, the order parameters of DNS-cytochrome b5 in DMPC vesicles increased on interaction with reductase at temperatures above the DMPC phase transition, and this increase was even more pronounced in cholesterol-containing vesicles, at temperatures from 15-30 degrees C. The enzyme activity of the DNS-cytochrome-b5 reductase system in DMPC vesicles was also greatly increased in the presence of cholesterol. These results show that interaction of vesicle-bound DNS-cytochrome b5 and NADH-cytochrome-b5 reductase leads to an increased degree of order of the dansyl-labeled cytochrome with little change in its rotational flexibility, and suggests that the increased order can be correlated with increased enzyme activity.     

On the origin of heterogeneity of fluorescence decay kinetics of reduced nicotinamide adenine dinucleotide

The fluorescence lifetimes of reduced nicotinamide adenine dinucleotide and other dihydronicotinamide derivatives were measured by picosecond laser excited time correlated single photon counting technique. All the dihydronicotinamide derivatives (including the simple model compound N-methyl-nicotinamide) had fluorescence decay profiles which could be fitted to double and triple exponentials in neutral aqueous solutions and in dimethyl sulfoxide respectively. It was concluded that the heterogeneity in the measured lifetimes arises from the inherent photoprocess of the dihydronicotinamide chromophore and not due to any intramolecular interaction as assumed in earlier studies. Some of the possible schemes for the fluorescence decay are discussed.     

On the choice of laser dyes for use in exciting tyrosine fluorescence decays

The choice of laser dyes for exciting tyrosine fluorescence using synchronously pumped cavity-dumped dye laser systems is discussed. Rhodamine 560 was found to be optimal for a system based on an argon-ion pumping laser, whereas rhodamine 575 was preferred using a frequency-doubled Nd:YAG laser. Modifications of our fluorescence decay instrument to permit rejection of multiphoton events using a microchannel plate photomultiplier are described. An example of a four-component resolution of tyrosine decays illustrates the dramatic resolution capabilities attainable.     

Distributions of fluorescence decay times for parinaric acids in phospholipid membranes

Analysis of fluorescence decay data for probes incorporated into model or biological membranes invariably requires fitting to more than one decay time even though the same probe exhibits nearly single-exponential decay in solution. The parinaric acids (cis and trans) are examples of this. Data are presented for both parinaric acid isomers in dimyristoylphosphatidylcholine membranes collected to higher precision than normally encountered, and the fluorescence decays are shown to be best described by a smooth distribution of decay times rather than by a few discrete lifetimes. The temperature dependence of the fluorescence decay reveals a clear shift in the distribution to longer lifetimes associated with the membrane phase transition at 23.5 degrees C. The physical significance is that fluorescence lifetime measurements appear to reflect a physical process with a distribution of lifetimes rather than several distinct physical processes.     

Constrained analysis of fluorescence anisotropy decay:application to experimental protein dynamics

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay.     

Spectral characteristics of fluorophores formed via interaction between all-trans-retinal with rhodopsin and lipids in photoreceptor membrane of retina rod outer segments

It is shown that all-trans-retinal under model conditions of its excessive accumulation in photoreceptor membranes interacts with amino groups of rhodopsin and lipids, forming at least three distinct fluorophores with fluorescence quantum yield 20–40 times higher than that of free all-trans-retinal. These retinal derivatives are likely precursors of photo- and cytotoxic fluorophores of lipofuscin and in particular of A2E. Spectral characteristics of fluorophores have been described. Picosecond time-resolved laser fluorescence spectroscopy was used to study kinetics of fluorescence decay of both free and bound all-trans-retinal; fluorophores were determined and their lifetimes have been measured. Based on calculations it is shown that the decay kinetics of all-trans-retinal derivatives consists of three components with lifetimes equal to 48, 208, and 900 ps; kinetics of free all-trans-retinal is monoexponential with lifetime of 31 ps. The chemical nature of fluorophores with the lifetimes obtained is discussed.     

Effect of quinone on the fluorescence decay dynamics of endogenous flavin bound to bacterial luciferase

The interaction of quinone with luciferase from Photobacterium leiognathi was studied based on the fluorescence decay measurements of the endogenous flavin bound to the enzyme. Homologous 1,4-quinones, 1,4-benzoquinone, methyl-1,4-benzoquinone, 2-methyl-5-isopropyl-1,4-benzoquine and 1,4-naphthoquinone, were investigated. In the absence of quinone, the fluorescence intensity and anisotropy decays of the endogenous flavin exhibited two intensity decay lifetimes (~ 1 and 5 ns) and two anisotropy decay lifetimes (~ 0.2 and 20 ns), suggesting a heterogeneous quenching and a rotational mobility microenvironment of the active site of the luciferase, respectively. In the presence of quinone, the intensity decay heterogeneity was largely maintained, whereas the fraction of the short anisotropy decay component and the averaged rotational rate of FMN increased with the increasing hydrophobicity of the quinone. We hypothesize that the hydrophobicity of the quinone plays a role in the non-specific inhibition mechanism of xenobiotic molecules in the bacterial bioluminescence system via altering the rotational mobility of the endogenous flavin in the luciferase.     

Thermal stability of a flavoprotein assessed from associative analysis of polarized time-resolved fluorescence spectroscopy

Upon gradually heating a particular mutant of the flavoprotein NADH peroxidase, it was found from the peculiar time-resolved fluorescence anisotropy pattern of the flavin prosthetic group (FAD) that, at elevated temperature, FAD is released from the tetrameric enzyme. Since in this case a mixture of free and enzyme-bound FAD contributes to the time-dependent fluorescence anisotropy, its analysis can only be accomplished by an associative fitting model, in which specific fluorescence lifetimes of both species are linked to specific correlation times. In this letter the general approach to the associative polarized fluorescence decay analysis is described. The procedure can be used for other flavoproteins to determine the temperature at which the onset of thermal denaturation will start, leading to release of the flavin prosthetic group. Received: 20 November 1998 / Revised version: 6 April 1999 / Accepted: 8 April 1999     

One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins

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.