A model of dynamic quenching of fluorescence in globular proteins

A model is presented for the quenching of a fluorophore in a protein interior. At low quencher concentration the quenching process is determined by the acquisition rate of quencher by the protein, the migration rate of quencher in the protein interior, and the exit rate of quencher from the protein. In cases where the fluorescence emission observed in the absence of quencher could be described by a single exponential decay, the presence of quencher led to doubly exponential decay times, and the aforementioned exit rates of the quencher could be determined from experimental data. At high quencher concentration, the processes became more complex, and the deterministic rate equations used at low quencher concentration had to be modified to take into account the Poisson distribution of quencher molecules throughout the protein ensemble and also by using a migration rate for quencher in the protein interior that is a function of the quencher concentration. Simulations performed for typical fluorescent probes in proteins showed good agreement with experiments.     

Viscosity dependence of acrylamide quenching of ribonuclease T1 fluorescence. The gating mechanism.

The structural regulation of the access of acrylamide molecules, as quenchers, to the buried tryptophans of a protein can be modelled by a simple gate concept. Such a gate, when open, allows transient exposure of the fluorophore to the quencher molecule in solution. We have previously shown that the observed viscosity dependence of acrylamide quenching process in ribonuclease T1 (RNAse T1) is not reconcilable with the gating mechanism. However, on that occasion, we neglected the effect of changes in the activity of the quencher molecule and the possible presence of static quenching. The experimental observation of a considerable contribution by static quenching and the realization that static quenching might produce dramatic effects in steady state measurements led us to reexamine the question. It is shown that in a gating model the static component can also influence the apparent dynamic quenching. In this paper, we present derived equations for the gated quenching mechanism including possible contributions from the static component. We also carefully remeasured the acrylamide quenching of RNAase T1 as a function of increasing glycerol concentration. Computer simulations were carried out to compare the experimental data set to the generalized model. We reach the conclusion that even the new, quite complex equations fail to predict the qualitative and quantitative features of the observed quenching experiments. We arrived at the conclusion that the fluorophore is never the target of the quencher molecules in solution.     

Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes.

The design and application of a recently developed type of fluorogenic substrates for proteolytic enzymes is reviewed. The substrates consist of peptide chains constructed to match the specificity of the particular enzyme and to bear a suitable chromophore at each side of the cleavable bond. One of the chromophores is a fluorescent group and the other is a quencher that causes a great reduction of fluorescence intensity of the fluorophore, either by direct intramolecular encounter or by radiationless resonance energy transfer. Enzymic cleavage of the molecule is followed by release of fluorescence as the result of cancelling the quenching interaction between the chromophores. The properties of such substrates and their possible future applications are discussed.     

A non-linear least-squares approach to the resolution of heterogeneous fluorescence from multitryptophan proteins

Protein heterogeneous fluorescence results from the different microenvironment of each emitting chromophore. The structural and dynamic information contained in this emission can be extracted to some extent by selective quenching experiments. In this work, graphical and numerical methods are described for the analysis of protein emission in terms of three separated contributions: a fluorescence fraction which is not accessible to the quencher and two additional fractions with different solvent exposure. ‘Static quenching’ deviations from Stern-Volmer behaviour are also discussed. The application of these methods is exemplified on simulated quenching experiments and real data on acrylamide quenching of lysozyme fluorescence.     

Fluorescence quenching of methyl-2-aminobenzoate in the presence of beta-cyclodextrin: A model for the dynamic quenching of chromophores in hydrophobic regions of biopolymers

The binding constant of methyl-2-aminobenzoate to beta-cyclodextrin was determined by fluorescence titration to be 92.1M^?1 at 25°C in pH 7 buffer. Beta-cyclodextrin dramatically protects methyl-2-aminobenzoate against quenching by iodate and protects, though much less efficiently, against the smaller quencher, iodide. The observed decrease in fluorescence lifetime of the methyl-2-aminobenzoate-beta-cyclodextrin complex on addition of quencher indicates that the quenching mechanism is collisional (dynamic). The dependence of quenching rate on solvent viscosity is less than expected from simple theoretical considerations. However, the extent of beta-cyclodextrin protection is essentially viscosity-independent. These model studies show the usefulness of iodate as a quencher and encourage further attempts at quantitative interpretation of quenching studies on chromophores attached to biopolymers.     

Compartmental analysis in photophysics: kinetics and identifiability of models for quenching of fluorescent probes in micelles

The parameters describing the kinetics of excited-state processes can possibly be recovered by analysis of the fluorescence decay surface measured as a function of the experimental variables. The identifiability analysis of a photophysical model assuming errorless time-resolved fluorescence data can verify whether the model parameters can be determined. In this work, we have used the methods of similarity transformation and Taylor series to investigate the identifiability of two models utilized to describe the time-resolved fluorescence quenching of stationary probes in micelles. The first model assumes that exchange of the quencher between micelles is much slower than the fluorescence decay of the unquenched probe (the 'immobile' quencher model). The second model assumes that quenchers exchange between the aqueous and micellar phases (the 'mobile' quencher model). For the 'immobile' quencher model, the rate constants for deactivation (k(0)) and quenching (k(q)) of the excited probe are uniquely identified together with the average number of quencher molecules per micelle. For the 'mobile' quencher model, the rate constants k(0) and k(q) are uniquely identified, as are the rate constants for entry (k(+)) and exit (k(-)) of one quencher molecule into and from a micelle, and the micellar aggregation number. The concomitant rate equations describing the time-resolved fluorescence are solved using z-transforms.     

Diffusion of small molecules through the structure of myoglobin. Environmental effects

The effect of the ambient solvent viscosity on the mobility of small molecules within myoglobin was studied by substituting Zn-protoporphyrin (ZnPP) for the native Fe-protoporphyrin and using it as an optical probe in the protein (ZnPPMb). The quenching of the ZnPPMb triplet state by oxygen, by anthraquinonesulfonate, and by methyl viologen was followed by exciting it with a laser flash and measuring its decay rate as a function of quencher concentration. The quenching rate constants were taken to measure the diffusion rate of the quencher within the protein. At room temperature, these constants were determined in aqueous and in 37% and 55% (by weight) glycerol-water solutions by measuring the ZnPPMb-delayed fluorescence at 606 nm. It was found that although the quenching rate constants varied the activation energies in the protein were very similar for the different quenchers. In aqueous solution, Ea = 6.0-7.4 kcal/mol; in 37% glycerol, Ea = 6.8-7.5 kcal/mol; and in 55% glycerol, Ea = 8.5-9.2 kcal/mol. The quenching rate of ZnPPMb by oxygen was also measured between 190K and 293K in 80% glycerol, and its triplet decay in the absence of oxygen was determined down to 120K in 88% glycerol. In all experiments, the quenching rates in the protein were compared to those of Zn-hematoporphyrin in the same solvent. The results are discussed in terms of Northrup and McCammon's gated reaction theory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen distribution and migration within Mbdes Fe and Hbdes Fe. Multifrequency phase and modulation fluorometry study.

Quenching of the intensity and lifetime of porphyrin fluorescence from Mbdes Fe and Hbdes Fe (iron-free myoglobin and hemoglobin) by oxygen was investigated using a multifrequency cross-correlation phase fluorometer. The single exponential decay characteristic of porphyrin emission of Mbdes Fe and Hbdes Fe became doubly exponential upon application of oxygen pressure. The results were interpreted in terms of a general model of dynamic quenching of fluorescence in globular proteins. The model accounted for the rate k+ of acquisition of quencher by the protein, the exit rate k- of quencher from the protein, and the migration rate chi of quencher in the protein interior. The values of k+, k-, and chi were different for Mbdes Fe and Hbdes Fe. The addition of 40% sucrose, which increased the bulk viscosity sixfold, modified these rates. These results are discussed and compared with previous quenching studies on proteins. The significance of these results and the model for the interpretation of protein quenching studies is emphasized.     

Quenching of fluorescence in membrane protein by hypocrellin B

The hypocrellin B (HB) was used as a fluorescence quencher to study the basic physical characteris-tics of HB in membrane systems, including the diffusion speed of quencher from aqueous phase into membrane phase, the partition coefficient (P) of quencher between membrane and water, and the fluorescence quenching constant of protein (Ksv; Kq). The experimental results show that the quenching of fluorescence in membrane protein by HB can be determined by the principle of dynamic quenching. The experimental process of fluorescence quenching was ob-served in detail by using the ESR technique. The signal of HB" was found to arise from an electron transfer from ex-cited trytophan to HB.     

Determination of the aggregation number of detergent micelles using steady-state fluorescence quenching.

The development of a simple, reliable method for determination of detergent micelle aggregation number that relies solely on measurement of steady-state fluorescence quenching is presented. The degree of steady-state fluorescence quenching of a micelle-solubilized fluorophore (pyrene) by a quencher that partitions greatly into the micelles (coumarin 153) is dependent on the micelle concentration, which can therefore be determined. The aggregation number is calculated as the micelle concentration/detergent monomer concentration (the total detergent concentration above the critical micelle concentration). For the determination to be accurate, the partition coefficient of the quencher into the micelle phase is determined and used to calculate the micellar concentration of quencher. Also, the quenching of pyrene by a coumarin 153 molecule within the same micelle must be complete, and this was confirmed by time-resolved fluorescence measurements. Aggregation numbers were determined for one cationic and several nonionic detergents and were found to be consistent with literature values. The approach presented is an improvement on a previous luminescence quenching technique (Turro, N.J., and A. Yekta. 1978. J. Am. Chem. Soc. 100:5951-5952) and can be used on cationic, anionic, and nonionic detergents with micelles ranging greatly in size and under varying conditions, such as detergent concentration, ionic strength, or temperature.     

Distance dependence of the tryptophan-disulfide interaction at the triplet level from pulsed phosphorescence studies on a model system.

In the present study the distance dependence of tryptophan-disulfide interaction is examined with a view to both utilizing the interaction as a more quantitative indicator of subtle conformational changes in proteins as well as elucidating the interaction mechanism. To examine perturbations specifically at the indole triplet level 2-(3-indolyl)-ethyl phenyl ketone (IEPK) in which excitation is transferred with high efficiency to the triplet state of the indole moiety was employed. Phosphorescence decays of IEPK excited by a laser pulse in 70/30 (vol/vol) ethanolether at 77 K were measured in the presence of various concentrations of simple disulfides. The nonexponential phosphorescence decays arising from a distribution of fixed chromophoreperturber separations and the steady-state quenching of IEPK were accounted for with an exponential dependence of the quenching rate constant with distance. The small effective Bohr radius (0.8 A) that appears in the exponent emphasizes the localized nature of the interaction. Comparison of the triplet quenching rate constant obtained at quencher contact with IEPK to that estimated in proteins suggests a dependence on the triplet energy of the indole moiety and an endothermic nature for the quenching process. The study predicts that in proteins tryptophan-disulfide interactions are very localized in nature and should give rise to detectable anomalous decays only out to 2 A beyond van der Waals contact between the interacting partners.     

Fluorescence quenching of the buried tryptophan residue of cod parvalbumin

Cod parvalbumin (isotype III) is a single tryptophan-containing protein. The fluorescence characteristics of this tryptophan residue (lambda em approximately 315 nm) suggest that it is buried from solvent and that it is located in an apolar core of the protein. Solute quenching studies of the tryptophan fluorescence of parvalbumin reveal dynamic quenching rate constants, kq, of 1.1 X 10(8) and 2.3 X 10(9) M-1 s-1 (at 25 degrees C) with acrylamide and oxygen, respectively, as quenchers. From temperature dependence studies, activation energies of 6.5 +/- 1.5 and 6.0 +/- 0.5 kcal/mol are found for acrylamide and oxygen quenching. The kq for acrylamide quenching is found to be relatively unchanged (+/- 10%) by an 8-fold increase in the bulk viscosity (glycerol/water mixture). These temperature and viscosity studies argue that the acrylamide quenching process involves a dynamic penetration of the quencher, facilitated by fluctuations in the protein's structure.     

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.

In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.     

Gated quenching of intrinsic fluorescence and phosphorescence of globular proteins. An extended model.

We present a theoretical model to account for the quenching data of macromolecular fluorescence and phosphorescence when the accessibility to the quencher is gated by a dynamic mechanism coupled to the fluctuation of the macromolecular matrix. We show that the model currently in use to interpret gated quenching processes gives only approximate results in both qualitative and quantitative terms, and it can be regarded as a specific case of the presented model. We show that the gating dynamics affect both the apparent accessibility (alpha obs) and Ksv values obtained by the modified Stern-Volmer plot. The effect of gating on alpha obs and Ksv depends upon the relative rate of gating compared to the excited state lifetime. The model allows us to predict the effect of viscosity on quenching if it takes place by a gated mechanism. The prediction can and is, in this case, compared to the existing data on glycerol effects on acrylamide quenching of the tryptophan fluorescence in RNAse T1. The result shows that a simple gated model is not compatible with the observed quenching behavior.     

Fluorescence characterization of type I collagen from normal and silicotic rats and its quenching dynamics induced by hypocrellin B

In this paper, we studied the quenching mechanism of intrinsic fluorescence of type I collagen by a new type photosensitizer and fluorescence quencher, hypocrellin B (HB). It was indicated that type I collagen can emit Tyr-intrinsic fluorescence with the excitation wavelength of Tyr (λ_ex = 269 nm). Its fluorescence decay conform to the triexponential rule of the fluorescence lifetime. The intrinsic fluorescence of type I collagen can be effectively quenched by HB through a process of charge and energy transference, which is involved in the collisional quenching, the dipolar inducement, and the formation of exciplex between HB and excited fluorophores of collagen. The fluorescence quenching would be weakened by higher ionic environments. The fluorescence emission and its quenching rate of abnormal silicotic collagen show falling trends, implying its much weakened potential of charge and energy transference, and its lessen bioelectric activities. In conclusion, the bioelectric properties of collagen depends on the perfect order of its molecular structure and orderly intramolecular and intermolecular interactions, which is important in its performing normal physiological functions. It is also demonstrated that the fluorescence quenching technique, using HB as a quencher, is truly an effectively method for biomolecular studies. © 1997 John Wiley & Sons, Inc. Biopoly 42: 219–226, 1997     

Organization and dynamics of pyrene and pyrene lipids in intact lipid bilayers. Photo-induced charge transfer processes.

The dynamics of fluorescence quenching and the organization of a series of pyrene derivatives anchored in various depths in bilayers of phosphatidylcholine small unilamellar vesicles was studied and compared with their behavior in homogeneous solvent systems. The studies include characterization of the environmental polarity of the pyrene fluorophore based on its vibronic peaks, as well as the interaction with three collisional quenchers: the two membrane-soluble quenchers, diethylaniline and bromobenzene, and the water soluble quencher potassium iodide. The system of diethylaniline-pyrene derivatives in the membrane of phosphatidylcholine vesicles was characterized in detail. The diethylaniline partition coefficient between the lipid bilayers and the buffer is approximately 5,800. Up to a diethylaniline/phospholipid mole ratio of 1:3 the perturbation to membrane structure is minimal so that all photophysical studies were performed below this mole ratio. The quenching reaction, in all cases, was shown to take place in the lipid bilayer interior and the relative quenching efficiencies of the various probe molecules was used to provide information on the distribution of both fluorescent probes and quencher molecules in the lipid bilayer. The quenching efficiency by diethylaniline in the lipid bilayer was found to be essentially independent on the length of the methylene chain of the pyrene moiety. These findings suggest that the quenching process, being a diffusion controlled reaction, is determined by the mobility of the diethylaniline quencher (with an effective diffusion coefficient D approximately 10(-7) cm2 s-1) which appears to be homogeneously distributed throughout the lipid bilayer. The pulsed laser photolysis products of the charge-transfer quenching reaction were examined. No exciplex (excited-complex) formation was observed and the yield of the separated radical ions was shown to be tenfold smaller than in homogenous polar solutions. The decay of the radical ions is considerably faster than the corresponding process in homogenous solutions. Relatively high intersystem crossing yields are observed. The results are explained on the basis of the intrinsic properties of a lipid bilayer, primarily, its rigid spatial organization. It is suggested that such properties favor ion-pair formation over exciplex generation. They also enhance primary geminate recombination of initially formed (solvent-shared) ion pairs. Triplet states are generated via secondary geminate recombination of ion pairs in the membrane interior. The results bear on the general mechanism of electron transfer processes in biomembranes.     

In vitro and in vivo investigation of chlorophyll binding sites involved in non‐photochemical quenching in Chlamydomonas reinhardtii

Non‐photochemical quenching (NPQ) of the light energy absorbed is one of the main photoprotective mechanisms evolved by oxygenic photosynthetic organisms to avoid photodamage, at a cost of reduced photosynthetic efficiency. Tuning of NPQ has been reported as a promising biotechnological strategy to increase productivity in both higher plants and unicellular microalgae. Engineering of NPQ induction requires the comprehension of its molecular mechanism(s), strongly debated in the last three decades with several different models proposed. In this work, the molecular details of NPQ induction was investigated at intramolecular level by in vitro and in vitro site‐specific mutagenesis on chlorophyll binding sites of the Light‐Harvesting Complex Stress‐Related 3 (LHCSR3) protein, the pigment binding complexes identified as the quencher during NPQ induction in the model organism for green algae Chlamydomonas reinhardtii. The results obtained demonstrate a correlation between the quenching activity of LHCSR3 variants in vitro and the NPQ phenotypes observed in vivo. In particular, multiple quenching sites in LHCSR3 cooperatively dissipating the excitation energy were revealed with a peculiar role of Chl 613, a chromophore located a close distance to carotenoid binding site L1.     

Fluorescence quenching of tryptophan by trifluoroacetamide

Trifluoroacetamide was found to be a good quencher of tryptophan fluorescence, and the quenching was shown to proceed via both a dynamic and a static process. The respective quenching constants were determined by the measurement of the decrease of the fluorescence lifetime in the presence of the quencher. The static and the bimolecular rate quenching constants of N-acetyltryptophanamide are equal to 0.34 1·mol^?1 and 1.9·10⁹ 1·mol^?1·s^?1, respectively. These values indicate that trifluoroacetamide is an efficient quencher of tryptophan fluorescence. This conclusion is also supported by a complete quenching of bovine serum albumin and wheat germ agglutinin fluorescence. In the case of lysozyme, trifluoroacetamide quenches the fluorescence of tryptophan residues which fluoresce with a maximum at 348 nm but not the buried tryptophan residues which fluoresce with a maximum at 333 nm. Trifluoroacetamide quenching of wheat germ agglutinin emission confirms the homogeneity and the high accessibility of emitting tryptophan residues, in agreement with a previous report (Privat, J.P. and Monsigny, M. (1975) Eur. J. Biochem. 60, 555–567). The tryptophan fluorescence decay of wheat germ agglutinin is biexponential even in the presence of the quencher; the static and bimolecular rate quenching constants are equal to 0.22 1·mol^?1 and 092·10⁹ 1·mol^?1·^?1, respectively. In the presence of a specific lectin ligand, the methyldi-N,N′-trifluoroacetyl-β- chitobioside, the quenching of wheat germ agglutinin fluorescence involves a direct contact between tryptophan residues and trifluoroacetamido groups of the ligand and in contrast with the quenching induced by free trifluoroacetamide shows that the tryptophan fluorescence is not fully quenched.     

Dynamic quenchers in fluorescently labeled membranes. Theory for quenching in a three-phase system.

The theory for quenching of fluorescently labeled membranes by dynamic quenchers is described for a three-phase system: a fluorescently labeled membrane, a nonlabeled membrane, and an aqueous phase. Two different experimental protocols are possible to determine quenching parameters. Using the first protocol, partition coefficients and bimolecular quenching constants were determined for a hydrophobic quencher in carbazole-labeled membranes in the presence of an unlabeled reference membrane. These parameters determined for 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) using this three-phase analysis were in good agreement with values determined by a two-phase analysis without the reference lipid. Hence, the theory was verified. In the second protocol, the quencher partition coefficient was determined for unlabeled membranes in the presence of a carbazole-labeled reference membrane. Partition coefficients for DDE determined by this method were the same as partition coefficients determined for carbazole-labeled membranes using the two-phase analysis. The greater ease in determining partition coefficients and bimolecular quenching constants by the three-phase analysis and, in particular, the ability to determine the partition coefficient in unlabeled membranes make the three-phase analysis especially useful. This method was used to study the effect varying the membrane lipid composition has on the partition coefficient. The data indicate that partition coefficients of DDE in fluid membranes are not dramatically dependent upon polar head group composition, fatty acid composition, or cholesterol content. However, partitioning into gel-phase lipids is at least 100-fold less than fluid-phase lipids.