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.     

Pressure dependence of fluorescence quenching reactions in proteins

The effect of hydrostatic pressure (0-2.6 kbar) on the acrylamide quenching of the fluorescence of indole derivatives and several single-tryptophan-containing proteins has been studied using phase fluorometry at 25 degrees C. For the model system, N-acetyl-L-tryptophanamide in water, there is essentially no pressure dependence of the quenching rate constant, kappa q. For the internal Trp residue of ribonuclease T1 and cod parvalbumin, there also is essentially no pressure dependence of the apparent kappa q at low pressure. Thus, the activation volume, delta V not equal to, for these quenching processes is approximately zero. Such small delta V not equal to values are expected for diffusion-limited reactions in water at this temperature. The low, apparent delta V not equal to values for the globular proteins characterize these quenching processes as involving very small amplitude fluctuations in the protein structures. Only for the poised tetramer in equilibrium monomer equilibrium of melittin were we able to observe a significant effect of pressure on kappa q and this is due to the pressure-induced shift in the equilibrium position.     

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.     

Diffusional barrier in the unfolding of a small protein

To determine how the dynamics of the polypeptide chain in a protein molecule are coupled to the bulk solvent viscosity, the unfolding by urea of the small protein barstar was studied in the presence of two viscogens, xylose and glycerol. Thermodynamic studies of unfolding show that both viscogens stabilize barstar by a preferential hydration mechanism, and that viscogen and urea act independently on protein stability. Kinetic studies of unfolding show that while the rate-limiting conformational change during unfolding is dependent on the bulk solvent viscosity, eta, its rate does not show an inverse dependence on eta, as expected by Kramers' theory. Instead, the rate is found to be inversely proportional to an effective viscosity, eta + xi, where xi is an adjustable parameter which needs to be included in the rate equation. xi is found to have a value of -0.7 cP in xylose and -0.5 cP in glycerol, in the case of unfolding, at constant urea concentration as well as under isostability conditions. Hence, the unfolding protein chain does not experience the bulk solvent viscosity, but instead an effective solvent viscosity, which is lower than the bulk solvent viscosity by either 0.7 cP or 0.5 cP. A second important result is the validation of the isostability assumption, commonly used in protein folding studies but hitherto untested, according to which if a certain concentration of urea can nullify the effect of a certain concentration of viscogen on stability, then the same concentrations of urea and viscogen will also not perturb the free energy of activation of the unfolding of the protein.     

Quenching of room temperature protein phosphorescence by added small molecules

A number of molecular agents that can efficiently quench the room temperature phosphorescence of tryptophan were identified, and their ability to quench the phosphorescence lifetime of tryptophan in nine proteins was examined. For all quenchers, the quenching efficiency generally follows the same sequence, namely, N-acetyltryptophanamide (NATA) greater than parvalbumin approximately lactoglobulin approximately ribonuclease T1 greater than liver alcohol dehydrogenase greater than aldolase greater than Pronase approximately edestin greater than azurin greater than alkaline phosphatase. Quenching rate constants for O2 and CO are relatively insensitive to protein differences, while H2S and CS2 are somewhat more sensitive. These small molecule agents appear to act by penetrating into the proteins. However, penetration to truly buried tryptophans is less favorable than previously suggested; in five proteins studied, quenching efficiency by O2 is 20-1000 times lower than for NATA, and up to 10(5) lower for H2S and CS2. Larger and more polar quenchers--including organic thiols, conjugated ketones and amides, and anionic species--were also studied. The efficiency of these quenchers does not correlate with quencher size or polarity, the quenching reaction has low energy of activation, and quenching rates are insensitive to solvent viscosity. These results indicate that the larger quenchers do not approach the buried tryptophans by penetrating into the proteins, even on the long phosphorescence time scale, and are also inconsistent with a mechanism in which quencher encounter with the tryptophan occurs in free solution, as in a protein-opening reaction. The results obtained suggest that the quenching process involves a long-range radiationless transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of heavy water on protein flexibility

The effects of heavy water (D(2)O) on internal dynamics of proteins were assessed by both the intrinsic phosphorescence lifetime of deeply buried Trp residues, which reports on the local structure about the triplet probe, and the bimolecular acrylamide phosphorescence quenching rate constant that is a measure of the average acrylamide diffusion coefficient through the macromolecule. The results obtained with several protein systems (ribonuclease T1, superoxide dismutase, beta-lactoglobulin, liver alcohol dehydrogenase, alkaline phosphatase, and apo- and Cd-azurin) demonstrate that in most cases D(2)O does significantly increase the rigidity the native structure. With the exception of alkaline phosphatase, the kinetics of the structure tightening effect of deuteration are rapid compared with the rate of H/D exchange of internal protons, which would then assign the dampening of structural fluctuations in D(2)O to a solvent effect, rather than to stronger intramolecular D bonding. Structure tightening by heavy water is generally amplified at higher temperatures, supporting a mostly hydrophobic nature of the underlying interaction, and under conditions that destabilize the globular fold.     

Accessibilities of the sulfhydryl groups of native and photooxidized lens crystallins: a fluorescence lifetime and quenching study

Fluorescence lifetime and acrylamide quenching studies on the N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS)-labeled sulfhydryl groups of bovine lens alpha-, beta H-, and gamma-crystallins were carried out to characterize the microenvironment of the sulfhydryls and changes produced by singlet oxygen mediated photooxidation. For the untreated proteins, the lifetimes of the major decay component of the fluorescence-labeled crystallins were 15.2, 14.4, and 13.0 ns, and the quenching rate constant, kq, values were 16.6 x 10(7), 26.9 x 10(7), and 32.7 x 10(7) M-1 s-1 for alpha-, beta H-, and gamma-crystallins, respectively. The results indicate that as the polarity of the sulfhydryl site increased (i.e., its lifetime decreased), its accessibility to collisional quenching by acrylamide also increased. The minor decay component of the fluorescence label was not significantly quenched by acrylamide for all three classes of crystallins. When the proteins were irradiated in the presence of methylene blue, in a system generating singlet oxygen, the kq value for acrylamide quenching of the major decay component of alpha-crystallin decreased to zero, while its lifetime decreased to 6 ns. Neither the lifetime nor the kq of alpha-crystallin recovered completely in the presence of the singlet oxygen quencher sodium azide. Light-induced binding of the photosensitizer methylene blue to the crystallins was observed by absorption spectroscopy. The bound photosensitizer partially quenches the fluorescence lifetime of the N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) label in irradiated alpha-crystallin. Further decrease in the lifetime occurs as a result of the singlet oxygen mediated conformational change. The results suggest that the fluorescence lifetime of the AEDANS is fully quenched in the irradiated alpha-crystallin and there is no further quenching by acrylamide. An increase in the fraction of the minor component of beta H-crystallin which was inaccessible to acrylamide quenching was observed after irradiation. There was no effect of irradiation on the kq for acrylamide quenching of the major component of the decay of AEDANS bound to beta H- or gamma-crystallins. Static quenching was found to contribute significantly to the steady-state quenching plots of the polar sulfhydryl sites of irradiated alpha-crystallin and of untreated and irradiated beta H- and gamma-crystallins, but it had no detectable role in the case of untreated alpha-crystallin. Fluorescence anisotropy of the AEDANS label bound to the crystallins was higher in the irradiated crystallins as compared with the controls.     

Fluorescence quenching of dimeric and monomeric forms of yeast hexokinase (PII): effect of substrate binding steady-state and time-resolved fluorescence studies

Fluorescence quenching studies on the PII isoenzyme of yeast hexokinase have been performed using charged as well as polar uncharged quenchers. In both 'open' (i.e. in the absence of glucose) and 'closed' (i.e. in the presence of glucose) forms of the enzyme, bimolecular quenching rate constant (kq) for acrylamide is significantly larger than that of KI, indicating that all the tryptophans are not fully exposed to the solvent. Overall accessibility of tryptophans towards KI was greater in the presence of glucose than in the absence of glucose. At high ionic strength, the value of bimolecular quenching rate constant (kq) for KI did not change suggesting that the average environment of the accessible tryptophan residue(s) is almost neutral. Quenching by KI is dynamic in nature. Accessibility of tryptophans towards acrylamide at concentration > or = 0.2 M was more in the 'open' form of the enzyme than that observed in the 'closed' form whereas at concentration < or = 0.2 M no significant difference in the extent of quenching was observed. It is reasonable to conclude that glucose induced conformational change leads some tryptophan residue(s) to be more exposed and at the same time some tryptophan residue(s) in the hydrophobic region become more buried. Dimeric and monomeric forms of the enzyme behave similarly towards the quenching by acrylamide. In the unfolded state, the accessibility of tryptophans was considerably higher for both the quenchers. Temperature dependent study and the fluorescence lifetime data indicate that the mechanism of quenching by acrylamide is primarily dynamic in nature.     

Pressure/temperature effects on protein flexibilty from acrylamide quenching of protein phosphorescence.

Pressure is an effective modulator of protein structure and biological function. The influence of hydrostatic pressure (相似文献   

Quenching by acrylamide and temperature of a fluorescent probe attached to the active site of Ribonuclease

The fluorescence properties of ribonuclease labelled at its active site with N-(iodoacetylamino)-ethyl-5-naphthylamine-1-sulfonic acid have been studied at different temperatures and in the presence of acrylamide. The rate constant for the quenching of the fluorescence of labelled ribonuclease by acrylamide is apparently not limited by the accessibility of the probe: similar values are obtained for the native and denatured states of the protein. Instead, acrylamide seems to be a rather inefficient quencher of this fluorescent group ((acetamidoamino) ethyl-5-naphtylamine-1-sulfonic acid), as shown by non-linear Stern-Volmer representations, biphasic decay kinetics, and a low value of the rate constant.The fluorescence intensity of the native state of the labelled protein is highly sensitive to temperature and exhibits a 20% decrease for an increase of temperature of from 10°C to 30°C, independent of solvent viscosity. This thermal quenching is specific for the native conformation and disappears when the protein is unfolded. When the fluorescence life-time of the label is shortened by addition of acrylamide, the effect of temperature becomes identical for native and unfolded structures. This suggests that the cause of the thermal quenching is the presence of conformational fluctuations within the native protein which apparently take place in the time range from 35 to 200 ns.Abbreviations used 1,5-IAEDANS N-(iodoacetylamino)ethyl-5-naphthylamine-1-sulfonic acid - AEDANS (acetamidoamine)-ethyl-5-naphthylamine-1-sulfonic acid - RNase bovine pancreatic ribonuclease - AEDANS-RNase RNase labelled with AEDANS - ME-AEDANS (hydroxyethylthioacetamido)ethyl-5-naphthylamine-1-sulfonic acid: the product of the reaction between 1,5-IAEDANS and -mercaptoethanol (Hudson and Weber 1973) - Gu-HCl guanidine hydrochloride     

Frequency domain measurements of the fluorescence lifetime of ribonuclease T1.

Using multifrequency phase/modulation fluorometry, we have studied the fluorescence decay of the single tryptophan residue of ribonuclease T1 (RNase T1). At neutral pH (7.4) we find that the decay is a double exponential (tau 1 = 3.74 ns, tau 2 = 1.06 ns, f1 = 0.945), in agreement with results from pulsed fluorometry. At pH 5.5 the decay is well described by a single decay time (tau = 3.8 ns). Alternatively, we have fitted the frequency domain data by a distribution of lifetimes. Temperature dependence studies were performed. If analyzed via a double exponential model, the activation energy for the inverse of the short lifetime component (at pH 7.4) is found to be 3.6 kcal/mol, as compared with a value of 1.0 kcal/mol for the activation energy of the inverse of the long lifetime component. If analyzed via the distribution model, the width of the distribution is found to increase at higher temperature. We have also repeated, using lifetime measurements, the temperature dependence of the acrylamide quenching of the fluorescence of RNase T1 at pH 5.5. We find an activation energy of 8 kcal/mol for acrylamide quenching, in agreement with our earlier report.     

Effect of protein dynamics upon reactions that occur in the heme pocket of horseradish peroxidase

Free base and Pd porphyrin derivatives of horseradish peroxidase show long-lived excited states that are quenched by the presence of the peroxidase inhibitor, benzhydroxamic acid. The relaxation times of the excited-state luminescence and the rates of the quenching reaction for these derivatives of peroxidase were monitored as a function of pH, temperature, and viscosity with the view of examining how protein dynamics affect the quenching reaction. As solvent viscosity increases, the rate decreases, but at the limit of very high viscosity (i.e., high glycerol or sugar glass) the quenching still occurs. A model is presented that is consistent with the known structure of the enzyme-inhibitor complex. It is considered that the inhibitor is held at an established position but that solvent-dependent and independent motions allow a limited diffusion of the two reactants. Since there is a steep dependence upon distance and orientation, the diffusion toward the favorable position for reaction enhances the reaction rate. The solvent viscosity dependent and independent effects were separated and analyzed. The importance of internal reaction dynamics is demonstrated in the observation that rigidity of solvent imposed by incorporating the protein into glass at room temperature allows the reaction to occur, while the reaction is inhibited at low temperature. The results emphasize that protein dynamics plays a role in determining reaction rates.     

Protein dynamics. A time-resolved fluorescence, energetic and molecular dynamics study of ribonuclease T1

Studies using time-resolved fluorescence depolarization were performed on the internal motion of Trp 59 of ribonuclease T1 (EC 3.1.27.3) in the free enzyme, 2'-GMP-enzyme complex and 3'-GMP-enzyme complex. The Trp 59 motion was also studied in the free enzyme using molecular dynamics simulations. Energetic analysis of activation barriers to the Trp 59 motion was performed using both the transition state theory and Kramers' theory. The activation parameters showed a dependence on solvent viscosity indicating the transition state approach in aqueous solution to be inadequate. When taking solvent viscosity contributions into account agreement between the transition state and Kramers' theories was obtained. The results indicate the three enzyme forms to have different conformations with the free enzyme and 3'-GMP-enzyme complex being similar. Comparison of the experimental and theoretical results showed a good agreement on the Trp 59 motion in the free enzyme. Trp 59 appears to vibrate rapidly, with a relaxation time of the order of 1 ps, within free space in the protein matrix and to have a slower motion, with a relaxation time of the order of 100 ps, which is related to breathing of the surrounding protein matrix. Molecular dynamics results indicate high mobility in regions of the enzyme involved in the interaction with the guanine base of the inhibitor or substrate while much lower mobility occurred in residues involved in the catalytic mechanism of ribonuclease T1.     

Some regularities of dynamic accessibility of buried fluorescent residues to external quenchers in proteins

The temperature and viscosity dependences of quenching of buried tryptophan residues of several proteins by external ionic (iodide) and neutral (acrylamide) quenchers have been studied. The effective quenching rate constant is shown to be proportional to the diffusion coefficient of the free solvent (water or 20 vol% glycerol). This fact supports the idea that the accessibility of buried chromophores of proteins to quenchers is based on the dynamic perturbations in protein structure (the dynamic accessibility). These structural perturbations are assumed to be governed by some diffusion-limited processes in the solvent surrounding the protein molecule.     

Conformational changes of beta-lactoglobulin in sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles. A fluorescence and CD study.

The effect of beta-lactoglobulin encapsulation in sodium bis(2-ethylhexyl) sulfosuccinate reverse micelles on the environment of protein and on Trp was analysed at different water contents (omega0). CD data underlined the distortion of the beta-sheet and a less constrained tertiary structure as the omega0 increased, in agreement with a concomitant red shift and a decrease in the signal intensity obtained in steady-state fluorescence measurements. Fluorescence lifetimes, evaluated by biexponential analysis, were tau1 = 1.28 ns and tau2 = 3.36 ns in neutral water. In reverse micelles, decay-associated spectra indicated the occurrence of important environmental changes associated with omega0. Bimolecular fluorescence quenching by CCl4 and acrylamide was employed to analyse alterations in the accessibility of the two Trp residues in beta-lactoglobulin, induced by changes in omega0. The average bimolecular quenching constant was found not to depend on omega0, confirming the insolubility of this quencher in the aqueous interface, while increases with omega0. The drastic decrease with omega0 of kq, associated with the longest lifetime kq2(CCl4), comparatively to the increase of kq2(acrylamide), emphasizes the location of beta-lactoglobulin in the aqueous interfacial region especially at omega0> or = 10. The fact that (omega0 = 30) > kq2(acrylamide) (water) also confirms the important conformational changes of encapsulated beta-lactoglobulin.     

Red-edge excitation fluorescence spectroscopy of proteins in reversed micelles

The dependence of fluorescence emission maxima ofl-tryptophan and single-tryptophan-containing proteins (ribonuclease T1, melittin, and parvalbumin) on excitation wavelength has been studied in reversed micelle systems of sodium bis(2-ethyl-1-oxyl) sulfosuccinate (AOT). No effect of fluorescence maximum shift for different excitation wavelengths is observed for ribonuclease T1, in which a single tryptophan residue is located in the nonrelaxating, nonpolar protein interior.l-Tryptophan and the rest of the studied proteins, which contain single tryptophan residues exposed to the solvent, exhibit the dipolar relaxational processes of partly immobilized water molecules in micelles. This effect depends on the molar H₂O/AOT ratio. Circular dichroism measurements prove that there have been no structural changes of the studied proteins in micellar systems. The results provide information about dynamic relaxational processes in proteins.     

Singlet oxygen quenching and the redox properties of hydroxycinnamic acids.

The singlet oxygen quenching rate constants (kq) for a range of hydroxycinnamic acids in acetonitrile and D2O solutions were measured using time resolved near infrared phosphorescence in order to establish their antioxidant activity. The magnitude of kq observed depends on both the nature of the substituent groups and solvent polarity. The variations in kq depend on the energy of the hydroxycinnamic acid/molecular oxygen charge transfer states, (O2delta- ...HCAdelta+). In D2O the values of kq range from 4x10(7) M(-1) s(-1) to 4x10(6) M(-1) s(-1) for caffeic acid and o-coumaric acid respectively. In acetonitrile, the charge transfer energy levels are raised and this is reflected in lower singlet oxygen quenching rate constants with a kq value of 5x10(6) M(-1) s(-1) for caffeic acid. The phenoxyl radical spectra derived from the hydroxycinnamic acids were determined using pulse radiolysis of aqueous solutions and the reduction potentials were found to range from 534 to 596 mV. A linear correlation is observed between reduction potential, and hence free energy for electron transfer, and log kq. These correlations suggest a charge transfer mechanism for the quenching of singlet oxygen by the hydroxycinnamic acids.     

Amplitude spectrum of structural fluctuations in proteins from the internal diffusion of solutes of increasing molecular size: a Trp phosphorescence quenching study

The accessibility of O(2), acrylamide, and four acrylamide derivatives of increasing molecular size {N-(hydroxymethyl)acrylamide, N,N'-methylene-bisacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, and 2-acrylamido-2-methyl-1-propanesulfonic acid} to buried Trp residues in four proteins, as determined by dynamic quenching of their phosphorescence emission, was utilized for probing the amplitude range of structural fluctuations in these macromolecules. The quenching rate constant of each solute, k(q), was determined (at 25 and -5 °C) for liver alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, azurin, and alkaline phosphatase. The results show that high-frequency small amplitude motions pervade the protein globular fold, permitting relatively unhindered diffusion of small diatomic molecules all the way to compact cores of the macromolecule. For larger solutes, the access to deep regions drops sharply with molecular size, with acrylamide probably representing a threshold for diffusion of a solute through homogeneous compact domains, on the long second time scale. The results emphasize the variability in the amplitude of protein motions between deep cores and more superficial regions of the globular fold and unveil the existence of unexpectedly large amplitude low-activation barrier fluctuations permitting the penetration of solutes with comparatively large M(w) values.     

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.     

Acrylamide fluorescence quenching studies on the actin-induced change in protein dynamics in the subfragment-1/subfragment-2 link region of cardiac myosin

In order to obtain information about the actin-induced conformational change around the subfragment-1/subfragment-2 link region of myosin, measurements of the fluorescence quenching by acrylamide were made on cardiac myosin and its heavy meromyosin, in which the reactive lysyl residue located in the link region was labeled with an extrinsic fluorophore, the N-methyl-2-anilino-6-naphthalenesulfonyl group. The results with the model compound indicated the involvement of a collisional quenching mechanism for the fluorophore. The quenching rate constant calculated from measured quenching constants using available lifetime data was extremely low for the labeled myosin (0.59 X 10(8) M-1 . S-1), suggesting that the fluorophore bound to myosin is surrounded by segments of proteins. This value was independent of the solvent viscosity, indicating that the quenching reaction is limited by fluctuations in the protein matrix, which produce the inward movement of acrylamide. Chymotryptic digestion of the labeled myosin, which yielded the light chain-2-deficient heavy meromyosin, made the bound fluorophore slightly exposed. Addition of F-actin resulted in about 40% reduction in the quenching rate constants for the labeled myosin and heavy meromyosin. The actin effect was reversed by adding ATP. These results suggest that the binding of actin to myosin makes the protein matrix around the subfragment-1/subfragment-2 link region less mobile.