Viscosity dependence of the solute quenching of the tryptophanyl fluorescence of proteins

We have studied the viscosity dependence of the acrylamide quenching of the fluorescence on the internal tryptophan residues in cod parvalbumin and ribonuclease T1, as well as the model systems. N-acetyl-L-tryptophanamide and glucagon. For the latter systems, the apparent rate constant, kq(app), for acrylamide quenching shows a typical diffusion-limited behavior. For parvalbumin and ribonuclease T1, however, the viscosity dependence of kq(app) is quite different. There is little change in the kq(app) values on increasing the bulk viscosity from 1 to 10 cP (by addition of glycerol), but a further increase from 10 to 100 cP results in a significant reduction in the kq(app). Both an unfolding mechanism and a quencher penetration mechanism are considered to explain the results. Only the penetration mechanism is found to be consistent, and our data are interpreted as indicating that the rate-limiting step for quenching goes from being that of diffusion through the protein matrix, at low viscosity, to diffusion through the bulk solvent, at high viscosity. By also considering the Kramers' relationship in fitting our data, we are able to obtain insight regarding the coupling between internal fluctuations in the structure of the protein and motion of the bulk solvent. For parvalbumin and ribonuclease T1, the internal dynamics are found to be very weakly coupled to the bulk.     

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.     

Pressure dependence of pyrene excimer fluorescence in human erythrocyte membranes

The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1–2080 bar) and temperature (5–40°C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19–21°C at 1 bar and 28–31°C at 450 bar, a shift with pressure of approx. 20–22 K per kbar. Corresponding transition temperatures of the liposomes were 21°C at 1 bar and 33°C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane.     

Penetration of dioxygen into proteins studied by quenching of phosphorescence and fluorescence

Experiments were done to measure the ability of dioxygen to collisionally quench the phosphorescent and fluorescent tryptophans in alcohol dehydrogenase and alkaline phosphatase. In all cases, luminescence is quenched with rate constants close to 1 x 10(9) M-1 s-1. The rate of reaching the buried tryptophans is little affected by solvent viscosity due to added glycerol. Quenching by dioxygen is not due to a protein-opening reaction. It appears to be rate limited by internal protein diffusion rather than at the entry step. Dioxygen appears to enter the proteins directly, as in liquidlike diffusion, rather than through transiently forming channels that are only present a small fraction of the time. A high-pressure oxygen system is described that considerably facilitates fluorescence quenching experiments.     

The reactivity of tryptophan residues in proteins. Stopped-flow kinetics of fluorescence quenching.

The quenching of tryptophan fluorescence by N-bromosuccinamide, studied by the fluorescence stopped-flow technique, was used to compare the reactivities of tryptophan residues in protein molecules. The reaction of N-bromosuccinamide with the indole group of N-acetyltryptophanamide, a model compound for bound tryptophan, followed second-order kinetics with a rate constant of (7.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1 at 23 degrees C. The rate does not depend on ionic strength or on the pH near neutrality. The non-fluorescent intermediate formed from N-acetyltryptophanamide on the reaction with N-bromosuccinamide appears to be a bromohydrin compound. The second-order rate constant for fluorescence quenching of tryptophan in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1. Apocytochrome c has the conformation of a random coil with the single tryptophan largely exposed to the solvent. The rate constant for the fluorescence quenching of the tryptophan in apocytochrome c by N-bromosuccinamide was (3.7 +/- 0.3) . 10(5) dm3 . mol-1 . s-1. The fluorescence quenching by N-bromosuccinamide of the tryptophan residues incorporated in alpha-chymotrypsin at pH 7.0 showed three exponential terms from which the following rate constants were derived: 1.74 . 10(5), 0.56 . 10(5) and 0.11 . 10(5) dm3 . mol-1 . s-1. This protein is known to have eight tryptophan residues in the native state, six residues at the surface, and two buried. Three of the surface tryptophans have the indole rings protruding out of the molecule and may account for the fastest kinetic phase of the quenching process. The intermediate phase may be due to three surface tryptophans whose indole rings point inwards, and the slowest to the two interior tryptophan residues.     

Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies.

Acrylamide is an efficient quencher of tryptophanyl fluorescence which we report to be very discriminating in sensing the degree of exposure of this residue in proteins. The quenching reaction involves physical contact between the quencher and an excited indole ring, and can be kinetically described in terms of a collisional and a static component. The rate constant for the collisional component is a kinetic measure of the exposure of a residue in a protein, and values ranging from 4 X 10(9) M-1 S-1 for the fully exposed tryptophan in the polypeptide, adrenocorticotropin, to less than 5 X 10(8) M-1 S-1 for the buried residue in azurin have been found. Static quenching is readily detected in proteins that are denatured, or contain only a single fluorophor. Quenching patterns for most multi-tryptophan containing proteins are difficult to analyze precisely, but qualitative information can, nevertheless, be extracted. Applications of this probing technique for monitoring protein conformational changes, such as the acid-induced expansion of human serum albumin, and inhibitor binding to enzymes, are presented. The value of this method lies in its ability to sense not only the steady-state exposure of a residue in a protein, but also its dynamic exposure.     

The association of acrylamide with proteins. The interpretation of fluorescence quenching experiments

The association properties of acrylamide with a number of proteins in aqueous solution have been investigated by a fluorescence-quenching method previously used in micelles and lipid bilayers (Blatt, E., Chatelier, R.C. and Sawyer, W.H. (1984) Chem. Phys. Lett. 108, 397-400). At pH 7.0, acrylamide partitions between the bulk aqueous phase and the proteins, human serum albumin, monellin and ovalbumin. Comparison with an earlier method of analysis (Sikaris, K.A., Thulborn, K.A. and Sawyer, W.H. (1981) Chem. Phys. Lipids 29, 23-36) confirms the data quantitatively. For human serum albumin at pH 2.2, acrylamide associates according to both partition and binding processes. Equilibrium dialysis experiments performed for the latter system verify that acrylamide associates with proteins.     

Non-photochemical quenching of fluorescence in cyanobacteria

The pathways of energy dissipation of excessive absorbed energy in cyanobacteria in comparison with that in higher plants are discussed. Two mechanisms of non-photochemical quenching in cyanobacteria are described. In one case this quenching occurs as light-induced decrease of the fluorescence yield of long-wavelength chlorophylls of the photosystem I trimers induced by inactive reaction centers: P700 cation-radical or P700 in triplet state. In the other case, non-photochemical quenching in cyanobacteria takes place with contribution of water-soluble protein OCP (containing 3′-hydroxyechinenone) that induces reversible quenching of allophycocyanin fluorescence in phycobilisomes. The possible evolutionary pathways of the involvement of carotenoid-binding proteins in non-photochemical quenching are discussed comparing the cyanobacterial OCP and plant PsbS protein. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 10, pp. 1385–1395.     

Direct observation of structure transition of donor-acceptor labeled peptides temperature dependence of fluorescence quenching kinetics

From the temperature dependence of fluorescence quenching kinetics, a novel temperature-sensitive structure transition was discovered in a donor-acceptor labeled peptides. Perfect agreement of related abrupt changes in a(1),tau(1) and tau(2) gives strong support for the existence of this structure transition, which suggests it is possible to develop peptides that can be rapidly and reversibly switched between two structure states in response to temperature shift.     

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.     

Distance mapping in proteins using fluorescence spectroscopy: the tryptophan-induced quenching (TrIQ) method

Studying the interplay between protein structure and function remains a daunting task. Especially lacking are methods for measuring structural changes in real time. Here we report our most recent improvements to a method that can be used to address such challenges. This method, which we now call tryptophan-induced quenching (TrIQ), provides a straightforward, sensitive, and inexpensive way to address questions of conformational dynamics and short-range protein interactions. Importantly, TrIQ only occurs over relatively short distances (～5-15 ?), making it complementary to traditional fluorescence resonance energy transfer (FRET) methods that occur over distances too large for precise studies of protein structure. As implied in the name, TrIQ measures the efficient quenching induced in some fluorophores by tryptophan (Trp). We present here our analysis of the TrIQ effect for five different fluorophores that span a range of sizes and spectral properties. Each probe was attached to four different cysteine residues on T4 lysozyme, and the extent of TrIQ caused by a nearby Trp was measured. Our results show that, at least for smaller probes, the extent of TrIQ is distance dependent. Moreover, we also demonstrate how TrIQ data can be analyzed to determine the fraction of fluorophores involved in a static, nonfluorescent complex with Trp. Based on this analysis, our study shows that each fluorophore has a different TrIQ profile, or "sphere of quenching", which correlates with its size, rotational flexibility, and the length of attachment linker. This TrIQ-based "sphere of quenching" is unique to every Trp-probe pair and reflects the distance within which one can expect to see the TrIQ effect. Thus,TrIQ provides a straightforward, readily accessible approach for mapping distances within proteins and monitoring conformational changes using fluorescence spectroscopy.     

Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77 K II. ATP-dependent quenching

In intact, uncoupled type B chloroplasts from spinach, added ATP causes a slow light-induced decline ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{min}$) of chlorophyll a fluorescence at room temperature. Fluorescence spectra were recorded after fast cooling to 77 K and normalized with fluorescein as an internal standard. Related to the fluorescence quenching at room temperature, an increase in Photosystem (PS) I fluorescence (F₇₃₅) and a decrease in PS II fluorescence (F₆₉₅) were observed in the low-temperature spectra. The change in the $\text{F}{}_{735}\text{F}{}_{695}$ ratio was abolished by the presence of methyl viologen. Fluorescence induction at 77 K of chloroplasts frozen in the quenched state showed lowered variable (F_v) and initial (F₀) fluorescence at 690 nm and an increase in F₀ at 735 nm. The results are interpreted as indicating an ATP-dependent change of the initial distribution of excitation energy in favor of PS I, which is controlled by the redox state of the electron-transport chain and, according to current theories, is caused by phosphorylation of the light-harvesting complex.     

Reversal of quinone-induced chlorophyll fluorescence quenching

We have used two methods to investigate the reversibility of the interaction of substituted quinones with the thylakoid membrane of plant chloroplasts. Treatment of chloroplasts with added quinones lowers the room-temperature Photosystem II chlorophyll fluorescence intensity by variable amounts depending on the identity and concentration of the quinone. The extent of restoration of the chlorophyll fluorescence level is used as a measure of the effectiveness of the reversal technique. One reversal method involves the addition of thiols to quinone-treated chloroplasts to alter the quinone in a chemical way via a nucleophilic 1,4-Michael addition. In general, the modified quinones exhibit a lower affinity for the thylakoid membrane, as evidenced by an accompanying increase in chlorophyll fluorescence. The thiol concentrations necessary for quenching reversal are found to be in the order [dithiothreitol] less than [2-mercaptoethanol] less than [glutathione]. The second reversal method examines the extent to which added quinones can be removed from thylakoid membranes using a concentration gradient established by resuspension of quinone-treated chloroplasts in quinone-free media. The results further support the reversible nature of the quinone inhibition and indicate that the extent of recovery is dependent upon the degree of fluorescence inhibition originally induced by the added quinone.     

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.     

Depth profiling in membranes by fluorescence quenching

Membrane penetration depth is an important parameter in relation to membrane structure and organization. A methodology has been developed to analyze the membrane penetration depths of fluorescent molecules or groups utilizing differential fluorescence quenching caused by membrane embedded spin-label probes located at different depths. The method involves determination of the parallax in the apparent location of fluorophores, detected when quenching by phospholipids spin-labelled at two different depths is compared. By use of relatively simple algebraic expressions, the method allows calculation of depth in å. This method has been used to determine the location of fluorophores in NBD-labelled lipids and anthroyloxy-labelled fatty acids in model membranes and of the membrane embedded tryptophan residues in the reconstituted nicotinic acetylcholine receptor.     

Nonphotochemical quenching of chlorophyll fluorescence in Chlamydomonas reinhardtii

Unlike plants, Chlamydomonas reinhardtii shows a restricted ability to develop nonphotochemical quenching upon illumination. Most of this limited quenching is due to state transitions instead of DeltapH-driven high-energy state quenching, qE. The latter could only be observed when the ability of the cells to perform photosynthesis was impaired, either by lowering temperature to approximately 0 degrees C or in mutants lacking RubisCO activity. Two main features were identified that account for the low level of qE in Chlamydomonas. On one hand, the electrochemical proton gradient generated upon illumination is apparently not sufficient to promote fluorescence quenching. On the other hand, the capacity to transduce the presence of a DeltapH into a quenching response is also intrinsically decreased in this alga, when compared to plants. The possible mechanism leading to these differences is discussed.