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.     

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.     

Effect of acrylamide on aldolase structure. I. Induction of intermediate states.

Acrylamide is a fluorescence quencher frequently applied for analysis of protein fluorophores exposure with the silent assumption that it does not affect the native structure of protein. In this report, it is shown that quenching of tryptophan residues in aldolase is a time-dependent process. The Stern-Volmer constant increases from 1.32 to 2.01 M-1 during the first 100 s of incubation of aldolase with acrylamide. Two tryptophan residues/subunit are accessible to quenching after 100 s of aldolase interaction with acrylamide. Up to about 1.2 M acrylamide concentration enzyme inactivation is reversible. Independent analyses of the changes of enzyme activity, 1ANS fluorescence during its displacement from aldolase active-site, UV-difference spectra and near-UV CD spectra were carried out to monitor the transition of aldolase structure. From these measurements a stepwise transformation of aldolase molecules from native state (N) through intermediates: I1, T, I2, to denatured (D) state is concluded. The maxima of I1, T, I2 and D states populations occur at 0.2, 1.0, 2.0 and above 3.0 M of acrylamide concentration, respectively. Above 3.5 M, acrylamide aldolase molecules become irreversibly inactivated.     

Vertical fluctuations of phospholipid acyl chains in bilayers

The possibility of vertical displacement of acyl chains in lipid bilayers has been examined by quenching the fluorescence of 2-(9-anthroyloxy)palmitic acid with 5- and 16-doxylstearates in dipalmitoylphosphatidylcholine unilamellar vesicles. Measurement of lifetime and steady-state quenching showed that the dynamic component of quenching was independent of the transverse position of the quencher indicating that a quencher at the 16-position could interact with a fluorophore at the 2-position with high frequency. The differences in steady-state quenching could be accounted for by the differences in the static component of quenching. The results provide further evidence for rapid vertical displacements of acyl chains in phospholipid bilayers.     

Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes

An important consideration in the design of oligonucleotide probes for homogeneous hybridization assays is the efficiency of energy transfer between the fluorophore and quencher used to label the probes. We have determined the efficiency of energy transfer for a large number of combinations of commonly used fluorophores and quenchers. We have also measured the quenching effect of nucleotides on the fluorescence of each fluorophore. Quenching efficiencies were measured for both the resonance energy transfer and the static modes of quenching. We found that, in addition to their photochemical characteristics, the tendency of the fluorophore and the quencher to bind to each other has a strong influence on quenching efficiency. The availability of these measurements should facilitate the design of oligonucleotide probes that contain interactive fluorophores and quenchers, including competitive hybridization probes, adjacent probes, TaqMan probes and molecular beacons.     

Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry

Site-specific fluorescence recordings have shown great promise in understanding conformational changes in signaling proteins. The reported applications on ion channels have been limited to extracellular sites in whole oocyte preparations. We are now able to directly monitor gating movements of the intracellular domains of cyclic nucleotide-gated channels using simultaneous site-specific fluorescence recording and patchclamp current recording from inside-out patches. Fluorescence signals were reliably observed when fluorophore was covalently attached to a site between the cyclic nucleotide-binding domain and the pore. While iodide, an anionic quencher, has a higher quenching efficiency in the channel's closed state, thallium ion, a cationic quencher, has a higher quenching efficiency in the open state. The state and charge dependence of quenching suggests movements of charged or dipolar residues near the fluorophore during CNG channel activation.     

Fluorescence-quenching study of percolation and compartmentalization in two-phase lipid bilayers.

Fluorescence quenching of a lipid-labeled fluorophore by a lipid spin-labeled quencher has been studied experimentally in two-component, two-phase phosphatidylcholine bilayers to examine the effect of phase connection and disconnection on quenching. Both fluorophore and quencher prefer the fluid phase. At the percolation threshold, the point at which the fluid phase becomes subdivided into may small disconnected domains, the quenching drops abruptly. This decrease in quenching is a function of the fluid-phase fraction and is due to the heterogeneous distribution of fluorophores and quenchers over the fluid-phase domains. Computer simulations of the system were carried out with a triangular lattice divided into closed compartments of variable size and reactant occupancy. The simulations demonstrate that the degree of quenching is reduced in the disconnected systems and that the reduction is correlated with the size of the disconnected domains. The combination of experimental data with simulations leads to the conclusion that at constant temperature the size of fluid-phase domains, nfluid, in the region of the coexistence of the fluid and gel phases is proportional to the fluid fraction, Xfluid. This is in a qualitative agreement with a previous electron spin resonance study of interlipid spin-spin interactions in the same two-component, two-phase bilayer system.     

The resolution of heterogeneous fluorescence of multitryptophan-containing proteins studied by a fluorescence-quenching method

From acrylamide quenching results, analyzed by an itterative non-linear least-squares method, we have shown that the fluorescence of multitryptophan-containing proteins, such as horse-liver alcohol dehydrogenase, 3-phosphoglycerate kinase and lysozyme, can be resolved for different segmental contributions, each characterized by collisional (Ki) and static (Vi) quenching constants. The ability to resolve the heterogeneous fluorescence of proteins makes it possible to follow changes in dynamics of the individual residues. In yeast 3-phosphoglycerate kinase, which contains only two tryptophan residues, three fluorescent fractions, characterized by different accessibility to the quencher, were observed. Two of them are assigned to one of the tryptophan residue. This may be interpreted in terms of conformational fluctuations, which facilitate the access of acrylamide molecules to the buried tryptophan residues.     

A photoreversible conformational change in 124 kDa Avena phytochrome

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I-, Cs+ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I-, was proven to be an ineffective quencher with Stern-Volmer constants, Ksv, of 0.60 and 0.63 M-1, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs+, showed about a 2-fold difference in the Ksv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25-37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs+ showed more than 40% increase in the Ksv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.     

Iodide penetration into lipid bilayers as a probe of membrane lipid organization.

The quenching efficiency of iodide as a penetrating fluorescence quencher for a membrane-associated fluorophore was utilized to measure the molecular packing of lipid bilayers. The KI quenching efficiency of tryptophan-fluorescence from melittin incorporated in DMPC bilayer vesicles peaks at the phase transition temperature (24 degrees C) of DMPC, whereas acrylamide quenching efficiency does not depend on temperature. The ability of iodide to penetrate the hydrocarbon region of the bilayer was examined by measuring the fluorescence quenching of the pyrene-phosphatidylcholine incorporated into DMPC vesicles (pyrene was attached to the 10th carbon of the sn-2 chain). The quenching efficiency of pyrene by iodide again shows a maximum at the lipid phase transition. We conclude that iodide penetrates the membrane hydrocarbon region at phase transition through an increased number of bilayer defects. The magnitude of change in quenching efficiency of iodide during lipid phase transition provides a sensitive technique to probe the lipid organization in membranes.     

Fluorescence quenching in lipid micelles and bilayers: Evaluation of bimolecular rate constants

Dynamic quenching of fluorophores and quenchers in lipid micelles and bilayers can yield information about the bimolecular rate constant for the quenching reaction, and hence information about the microviscosity of the fluorophore-quencher environment. When the fluorophore and quencher have relatively fixed transverse positions in the bilayer, the analysis of Sikaris et al. (Chem. Phys. Lipids. 29 (1981) 23) can be used to separate the microviscosity and proximity contributions to quenching. We now extend this method to show explicitly the effect of static quenching on the analysis. We show by simulation and experiment that a correction factor must be included when static quenching contributes to the observed quenching efficiency.     

Spatial proximity of ATP-sensitive tryptophanyl residue(s) and Cys-697 in myosin ATPase.

The reactive thiol Cys-697 (SH2) in myosin ATPase was labeled with a fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) (Hiratsuka, T. (1992) J. Biol. Chem. 267, 14941-14948). Although the tryptophan fluorescence of myosin subfragment-1 (S-1) was slightly affected by incorporation of the MIANS fluorophore, the tryptophan fluorescence of the resultant S-1 derivative (MIANS-S-1) was enhanced by ATP in a manner similar to that of unlabeled S-1. The quenching of tryptophan fluorescence of MIANS-S-1 was shown to result from a transfer of the excitation energy from tryptophanyl residue(s) to the MIANS fluorophore attached to SH2, which absorbed and fluoresced maximally at 325 and 418 nm, respectively. The energy transfer measurements were performed in the presence of acrylamide and compared to those performed in the absence of the quencher. The energy transfer efficiencies were found to be unaltered by acrylamide, indicating that the observed fluorescence energy transfer is originated exclusively from the tryptophanyl residue(s) that are not affected by acrylamide, i.e. the ATP-sensitive tryptophanyl residue(s) of S-1 (Torgerson, P. M. (1984) Biochemistry 23, 3002-3007). The distance between the tryptophanyl residue(s) and Cys-697 was calculated to be 27 A assuming a single donor-acceptor pair. Trp-510 is proposed to be one of the ATP-sensitive tryptophanyl residues.     

A photoreversible conformational change in 124 kDa Avena phytochrome

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I⁻, Cs⁺ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I⁻, was proven to be an ineffective quencher with Stern-Volmer constants, K_sv, of 0.60 and 0.63 M⁻¹, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs⁺, showed about a 2-fold difference in the K_sv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25–37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs⁺ showed more than 40% increase in the K_sv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.     

Protein fluorescence quenching by small molecules: protein penetration versus solvent exposure

Experiments were done to test the thesis that acrylamide and similar small molecules can penetrate into proteins on a nanosecond time scale. The approach taken was to measure the pattern of fluorescence quenching exhibited by quenching molecules differing in molecular character (size, polarity, charge) when these are directed against protein tryptophans that cover the whole range of tryptophan accessibility. If quenching involves protein penetration and internal quencher migration, one expects that larger quenchers and more polar quenchers should display lesser quenching. In fact, no significant dependence on quencher character was found. For proteins that display measurable quenching, the disparate quenchers studied display very similar quenching rate constants when directed against any particular protein tryptophan. For several proteins having tryptophans known to be buried, no quenching occurs. These results are not consistent with the view that the kinds of small molecules studied can quite generally penetrate into and diffuse about within proteins at near-diffusion-limited rates. Rather the results suggest that when quenching is observed, the pathway involves encounters with tryptophans that are partially exposed at the protein surface. Available crystallographic results support this conclusion.     

Effects of Quenching Mechanism and Type of Quencher Association on Stern-Volmer Plots in Compartmentalized Systems

Fluorescence quenching techniques have been used extensively in recent years to examine reaction rates and the compartmentalization of components in lipid micelles and membranes. Steady-state fluorescence methods are frequently employed in such studies but the interpretation of the resulting Stern-Volmer plots is often hampered by uncertainties regarding the mode of association of the quencher with the lipid structure and the nature of the quenching mechanism. This paper presents a method for simulating steady-state Stern-Volmer plots in two phase systems, and shows how the forms of such plots are influenced by the type of association of the quencher with the membrane or micelle (partition and/or binding) and by the type of quenching mechanism (dynamic and/or static). Comparisons of simulated plots with experimental data must take into account the possible combinations of quencher association(s) and quenching mechanism(s). The methods presented are applicable to synthetic and natural membranes and provide a basis for comparing the quenching of fluorescent molecules in biological membranes of differing composition.     

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.     

A study of tryptophan fluorescence quenching of bifunctional alginate lyase from a marine bacterium Pseudoalteromonas sp. strain No. 272 by acrylamide

A fluorescence quenching study of a sole tryptophan residue of a bifunctional alginate lyase from Pseudoalteromonas sp. strain No. 272 was done in the presence and absence of substrates, oligomeric guluronic and its C5 isomer mannuronic acid, by a Stern-Volmer plot with a quencher, acrylamide. N-Acetyltryptophanamide and reduced and carboxymethylated alginate lyase showed large quenching constants, on the other hand, the native enzyme had small constants regardless of the presence or absence of the substrates. The result suggests that the tryptophan residue is located in a buried region of the enzyme molecule, but is barely accessible to acrylamide, and that the residue is not masked by the substrates with various degrees of polymerization.     

Detection of PCR products using self-probing amplicons and fluorescence.

Molecular diagnostics is progressing from low-throughput, heterogeneous, mostly manual technologies to higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays, and a number of techniques rely on energy transfer between a fluorophore and a proximal quencher molecule. In these methods, dual-labeled probes hybridize to an amplicon and changes in the quenching of the fluorophore are detected. We describe a new technology that is simple to use, gives highly specific information, and avoids the major difficulties of the alternative methods. It uses a primer with an integral tail that is used to probe an extension product of the primer. The probing of a target sequence is thereby converted into a unimolecular event, which has substantial benefits in terms of kinetics, thermodynamics, assay design, and probe reliability.     

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.     

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.