New trends in photobiology

Somogyi, B; Lakos, Zsuzsa

doi:10.1016/1011-1344(93)80035-8

Cited by 24 publications

(9 citation statements)

References 64 publications

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“…[1][2][3][4][5] Furthermore, quencher accessibility has been useful for revealing changes in the conformation of the polypeptide but, more importantly, also for investigating on the nature of its dynamical structure, that is, the frequency and amplitude of structural fluctuations that permit diffusion of solutes through generally well-packed and extensively bonded internal regions of the globular fold. 2,[6][7][8][9][10][11] Quenching studies measure the luminescence lifetime (τ) as a function of quencher concentration, [Q], and evaluate the bimolecular quenching rate constant, k q , from the gradient of the Stern-Volmer plot, where τ 0 is the unperturbed lifetime. With chromophores free in solution and efficient quenching reactions, k q is practically identical to the diffusion-limited rate constant k d , where k d ) 4πr 0 D (r 0 is the sum of molecular radii and D is the sum of the diffusion coefficients).…”

Section: Introductionmentioning

confidence: 99%

“…Quenching of Trp fluorescence and phosphorescence in proteins by added small solutes is widely employed in structural studies, for it can provide information on the location of Trp residues with respect to the aqueous phase and on the permeability of proteins to ligands of various molecular sizes. − Furthermore, quencher accessibility has been useful for revealing changes in the conformation of the polypeptide but, more importantly, also for investigating on the nature of its dynamical structure, that is, the frequency and amplitude of structural fluctuations that permit diffusion of solutes through generally well-packed and extensively bonded internal regions of the globular fold. ,− …”

Section: Introductionmentioning

confidence: 99%

“…Ambiguities with the interpretation of acrylamide F k q in terms of hindered diffusion may arise both from uncertainties with regard to the quenching mechanism and from potential artifacts linked to the propensity of acrylamide to bind to proteins. , Binding may result in partitioning of acrylamide into proteins and, in worst cases, may even cause perturbations of the native structure. , With regard to the mechanism, the recent demonstration that an effective interaction between acrylamide and the fluorescent state of indole does not require physical contact between the reactants points out that superficially buried Trps, namely within 4−5 Å of the aqueous interface, can be directly quenched by acrylamide in the solvent. In this respect, it may be significant that fluorescence quenching by acrylamide has not been observed in protein with more deeply buried Trp residues.…”

Section: Introductionmentioning

confidence: 99%

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Acrylamide Quenching of Protein Phosphorescence as a Monitor of Structural Fluctuations in the Globular Fold

1998

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This study examines acrylamide quenching of tryptophan room-temperature phosphorescence in proteins and the role that factors such as long-range interactions and environment-dependent quenching efficiency might play in the interpretation of bimolecular quenching rate constants in terms of hindered quencher migration through the globular fold. The distance dependence of the through-space quenching rate is evaluated by studying the effects of acrylamide on the phosphorescence intensity and decay kinetics of the indole analogue 2-(3-indoyl)ethyl phenyl ketone in propylene glycol/buffer glasses, at 120 K. Both steady-state and kinetic data are satisfactorily fitted by an exponential distance dependence of the rate, k(r) = k 0 exp[−(r − r 0)/r e], with a contact rate k 0 = 1.2 × 108 s-1 and an attenuation length r e = 0.29 Å. For a phosphorescence lifetime of 5 s, this rate yields an average interaction distance of 10 Å. The rate is temperature dependent, with k 0, estimated from the bimolecular quenching rate constant (P k q) of Trp analogues in liquids, increasing by about 10-fold from 120 to 293 K. Solvent effects on the quenching efficiency are tested with Trp analogues in water, propylene glycol, and dioxane. The quenching efficiency per collisional encounter is about 0.20 for water, 0.35 for propylene glycol, and drops to 0.025 in the aprotic, least polar dioxane. Acrylamide quenching rate constants are determined for a series of proteins and for experimental conditions appositely selected to test the importance of factors such as the degree of Trp burial and structural rigidity. Relative to P k q = 1.5 × 109 M-1 s-1 for Trp in the solvent, the magnitude of P k q for protected Trp residues in proteins ranges from a maximum of 6 × 104 M-1 s-1, for the most superficial W59 of RNase T1, to 10-1 M-1 s-1 for the most internal W109 of alkaline phosphatase. For most proteins, theoretical estimates of P k q based on the distance dependence of the rate exclude any quenching contribution from through-space interactions by acrylamide in the solvent. This finding, together with a clear correlation between P k q and other indicators of molecular flexibility, implies that in the millisecond-second time scale of phosphorescence acrylamide can migrate through the macromolecule and that its rate is a measure of the frequency and amplitude of the structural fluctuations underlying diffusional jumps. The origin of the discrepancy between fluorescence and phosphorescence quenching rates in proteins is discussed, and an alternative interpretation of fluorescence quenching data is provided.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Acrylamide Quenching of Protein Phosphorescence as a Monitor of Structural Fluctuations in the Globular Fold

1998

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“…Fluorescence quenching of aromatic and heteroaromatic hydrocarbons by aromatic amines, − aromatic nitriles, haloalkanes, − and halide ions − as well as by nitroxides , and oxygen , has been studied to determine the bimolecular rate constants, to characterize the solvent effects on quenching, and to determine the mechanisms of interaction between the fluorophore and quencher molecules. Moreover, collisional quenching of tryptophan fluorescence by a variety of ionic and neutral quenchers has been used to study the structure and dynamics of proteins − and membranes. , Collisional or dynamic quenching requires contact between the fluorophore and quencher molecules during the lifetime of the excited state. The quenching data can reveal information about accessibility of fluorophores in macromolecules to externally added quenchers and the diffusion of quenchers within proteins and membranes.…”

Section: Introductionmentioning

confidence: 99%

Distance-Dependent Fluorescence Quenching ofp-Bis[2-(5-phenyloxazolyl)]benzene by Various Quenchers

et al. 1996

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We report results of frequency-domain and steady-state measurements of the fluorescence quenching of p-bis-[2-(5-phenyloxazolyl)]benzene (POPOP) when quenched by bromoform (CHBr 3 ), methyl iodide (CH 3 I), potassium iodide (KI), 1,2,4-trimethoxybenzene (TMB), or N,N-diethylaniline (DEA). The quenching efficiency of these compounds decreased in the order DEA, TMB, KI, CH 3 I, CHBr 3 . In the case of DEA and TMB the measurements clearly confirm the applicability of the exponential distance-dependent quenching (DDQ) model, in which the bimolecular quenching rate k(r) depends exponentially on the fluorophore-quencher separation r, k(r) ) k a exp[-(r -a)/r e ], where a is the distance of closest approach. Simultaneous analysis of the frequency-domain and steady-state data significantly improved resolution of the recovered molecular parameters k a and r e . The data for DEA and TMB cannot be satisfactorily fit using either the Smoluchowski or CollinsKimball radiation boundary condition (RBC) model. The quenching behavior of the less efficient quenchers KI, CH 3 I, and CHBr 3 can be adequately described with both the DDQ and RBC models, but this may be a simple consequence of less efficient quenching. The efficiency of quenching is discussed on the basis of the mechanisms of interaction between the fluorophore and quencher molecules, which involves electron transfer and/or heavy atom effects. IntroductionCollisional quenching of fluorescence has been widely used in physical chemistry and biochemistry. Fluorescence quenching of aromatic and heteroaromatic hydrocarbons by aromatic amines, 1-13 aromatic nitriles, 14 haloalkanes, 15-24 and halide ions 25-27 as well as by nitroxides 28,29 and oxygen 30,31 has been studied to determine the bimolecular rate constants, to characterize the solvent effects on quenching, and to determine the mechanisms of interaction between the fluorophore and quencher molecules. Moreover, collisional quenching of tryptophan fluorescence by a variety of ionic and neutral quenchers has been used to study the structure and dynamics of proteins 32-40 and membranes. 41,42 Collisional or dynamic quenching requires contact between the fluorophore and quencher molecules during the lifetime of the excited state. The quenching data can reveal information about accessibility of fluorophores in macromolecules to externally added quenchers and the diffusion of quenchers within proteins and membranes.For many fluorophores in solution, including aromatic and heteroaromatic fluorophores, the intensity decays are often monoexponential. 43 However, in the presence of collisional quenching the monoexponential intensity decays become nonexponential due to transient effects in diffusion. 21,[44][45][46] This effect in collisional quenching of fluorescence is due to a rapid decay of closely spaced fluorophore-quencher pairs, followed by slower diffusion-limited quenching of remaining fluorophores. These effects can be readily detected by using frequency-domain fluorometry. [47][48][49] We have shown for several fl...

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“…A more precise explanation (Somogyi and Lakos, 1993) takes into consideration the part of fluorescence actually involved in gating, from a calculation of the probability that the excited fluorophore will not be quenched, on the one hand, and the possible participation of a static quenching, on the other hand. From the general point of view, the gating mechanism has received attention because it is consistent with the absence of a relationship between the quencher efficiency and the molecular size.…”

Section: Bousquet and Ettnermentioning

confidence: 99%

A possible tertiary structure change induced by acrylamide in the DNA-binding domain of the Tn10-encoded Tet repressor. A fluorescence study

Bousquet¹,

Ettner

1996

J Protein Chem

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A thorough investigation of the acrylamide fluorescence quenching of F75TetR, a mutant of the Tn10-encoded TetR repressor containing a single Trp residue at position 43, was carried out. The Trp-43 residue is located in a helix alpha-turn-helix alpha (H-t-H) motif involved in the specific binding of F75TetR to the operator site in specific DNA. Distinct Ranges of acrylamide concentration have been assumed. At acrylamide concentrations below 0.15-0.2 M (a usual range of values in fluorescence quenching studies) the observed limited tertiary structure change induced by acrylamide is consistent with a noncooperative local unfolding of the DNA-binding domain. It is suggested that penetration of the neutral quencher could cause the deletion of a hydrophobic tertiary structure contact, partly involving TrP-43, responsible for the anchoring of the H-t-H motif inside the three-helix protein bundle, characterizing the N-terminal part. Correspondingly, the affinity of the mutant repressor for the operator was shown to decrease substantially (about five orders of magnitude), seemingly losing its specificity. A subsequent phase, up to 0.8 M acrylamide, was observed in which the involved intermediate protein structure is not further perturbed, nor is DNA binding.

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New trends in photobiology

Cited by 24 publications

References 64 publications

Acrylamide Quenching of Protein Phosphorescence as a Monitor of Structural Fluctuations in the Globular Fold

Acrylamide Quenching of Protein Phosphorescence as a Monitor of Structural Fluctuations in the Globular Fold

Distance-Dependent Fluorescence Quenching ofp-Bis[2-(5-phenyloxazolyl)]benzene by Various Quenchers

A possible tertiary structure change induced by acrylamide in the DNA-binding domain of the Tn10-encoded Tet repressor. A fluorescence study

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