Fluorescence‐quenching‐resolved spectra of fluorophores in mixtures and micellar solutions

Wasylewski, Zygmunt; Kaszycki, Paweł; Guz, Andrzej; Stryjewski, Wiesław

doi:10.1111/j.1432-1033.1988.tb14472.x

Cited by 16 publications

(11 citation statements)

References 18 publications

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“…Experimentally, we verified the linear dependence of the inverse of the lifetime with the concentration of quencher and obtain a bimolecular quenching rate constant of 1.89 3 10 9 s 21 mol L 21 conform to the results reported by Wasylewski et al (Wasylewski et al, 1988) (data not shown).…”

Section: Results and Discussion Calibration And Optimization Of The Isupporting

confidence: 90%

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Leray

Riquet

Richard

et al. 2008

Microscopy Res & Technique

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Frequency-domain fluorescence lifetime imaging microscopy (FLIM) has become a commonly used technique to measure lifetimes in biological systems. However, lifetime measurements are strongly dependent on numerous experimental parameters. Here, we describe a complete calibration and characterization of a FLIM system and suggest parameter optimization for minimizing measurement errors during acquisition. We used standard fluorescent molecules and reference biological samples, exhibiting both single and multiple lifetime components, to calibrate and evaluate our frequency domain FLIM system. We identify several sources of lifetime precision degradation that may occur in FLIM measurements. Following a rigorous calibration of the system and a careful optimization of the acquisition parameters, we demonstrate fluorescence lifetime measurements accuracy and reliability. In addition, we show its potential on living cells by visualizing FRET in CHO cells. The proposed calibration and optimization protocol is suitable for the measurement of multiple lifetime components sample and is applicable to any frequency domain FLIM system. Using this method on our FLIM microscope enabled us to obtain the best fluorescence lifetime precision accessible with such a system.

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Section: Results and Discussion Calibration And Optimization Of The Isupporting

confidence: 90%

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Leray

Riquet

Richard

et al. 2008

Microscopy Res & Technique

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“…In the GSCH model the solvent‐exposed conformer ( λ max ≈350 nm) is preferentially quenched over the buried conformer. Individual emission spectra from the conformers can be estimated by analyzing Stern–Volmer plots over the entire emission wavelengths [14]. A global analysis of quenching data according to Eq.…”

Section: Resultsmentioning

confidence: 99%

Dielectric relaxation in a single tryptophan protein

et al. 2001

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Although dielectric relaxation can significantly affect the intrinsic fluorescence properties of a protein, usually it is fast compared to fluorescence timescales and needs to be slowed down by adding viscogens or lowering temperature before its impact on fluorescence can be studied. We report here a remarkable blue shift in fluorescence upon bimolecular quenching in the single-tryptophan thermostable protein Bj2S, the 2S seed albumin from Brassica juncea, at ambient temperature and viscosity. The magnitude of the blue shift ( approximately 5 nm at 50% quenching by acrylamide) is striking in a single-tryptophan protein and is attributed to a slowly relaxing dielectric environment in Bj2S from red edge excitation, steady-state polarization and time-resolved fluorescence experiments. Our results have important implications on interpretation of fluorescence of proteins with highly constrained backbones and in designing model systems for studying slow protein solvation dynamics using Trp fluorescence as the reporter probe.

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“…The heterogeneous fluorescence emission of tryptophan residues in proteins can be resolved if there is a difference in their lifetime [5-71, anisotropy [8] or accessibility to an external quencher [9 -111. The fluorescence emission spectra of several twotryptophan-containing proteins have been separated into components using time-domain [4] or frequency-domain techniques [12].Recently [13,14] we have proposed a powerful method for the determination of fluorescence-quenching resolved spectra (FQRS). In this method the possibility of resolving the fluorescence emission spectra depends on differences in the dynamic Stern-Volmer quenching constant, K, of the component.…”

mentioning

confidence: 99%

“…Recently [13,14] we have proposed a powerful method for the determination of fluorescence-quenching resolved spectra (FQRS). In this method the possibility of resolving the fluorescence emission spectra depends on differences in the dynamic Stern-Volmer quenching constant, K, of the component.…”

mentioning

confidence: 99%

“…Since the K constant for an efficient quencher depends both on the fluorescence lifetime, T~, and the bimolecular rate quenching constant, k,, the heterogeneous fluorescence emission can be resolved into components if particular fluorophores differ in T~ and/or k , [I 31. Contrary to fluorescence lifetime measurements, this method allows the decomposition of overlapping spectra even if the components have similar or the same fluorescence lifetimes, but differ in bimolecular rate quenching constants [14]. Below, we report on measurements of the red-edge effect in several two-tryptophan-containing proteins.…”

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confidence: 99%

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Red‐edge excitation fluorescence measurements of several two‐tryptophan‐containing proteins

Wasylewski

Kołoczek

Waśniowska

et al. 1992

European Journal of Biochemistry

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The dependence of the fluorescence emission maximum of the tryptophan residues in several twotryptophan-containing proteins (horse liver alcohol dehydrogenase, yeast 3-phosphoglycerate kinase, Staphylococcus aureus metalloprotease and bee venom phospholipase A,) on the excitation wavelengths has been studied. Using fluorescence-resolved spectroscopy, we have dissected the contributions of particular tryptophan residues located in different parts of the protein molecule. The results demonstrate that dipolar structural relaxation can occur in the environment of tryptophan residues buried within protein molecules. The observed spectral shifts upon red-edge excitation of these residues can depend on temperature or ligand binding, as demonstrated in case ofmetalloprotease and alcohol dehydrogenase. No spectral shifts upon red-edge excitation have been observed for tryptophan residues totally exposed to the rapidly relaxing aqueous solvent.The intrinsic fluorescence emission of tryptophan residues in proteins provides a sensitive probe of the dynamic properties of their microenvironment. A variety of fluorescence methods, including time-and phase-resolved spectroscopy, have been used in such studies. Some information about protein dynamics can be obtained on the basis of steady-state fluorescence measurements. The steady-state collisional quenching of tryptophan residues buried inside a protein molecule reveals some information about the protein dynamics, since a contact between a fluorophore and a quencher is required in quenching reactions. Other dynamic properties of proteins, which can also be deduced from steady-state measurements, are associated with the phenomenon called the rededge excitation effect [I]. In this phenomenon the tryptophan fluorescence emission spectra can shift to the longer wavelengths as the excitation wavelength is increased toward the red side of the tryptophan absorption.Recently Demchenko [2] has shown that, in several singletryptophan-containing proteins, the red-edge effect is particularly visible in proteins which show fluorescence emission maximum at about 325 -340 nm. In the case of short-wavelength-emitting proteins like azurin, parvalbumin and ribonuclease T I and proteins emitting at a long wavelength (about 350 nm), like melittin in water, no spectral shifts upon the rededge excitation have been observed [2]. This might be due to the fact that in azurin, parvalbumin and ribonuclease TI the single tryptophan residues are located in a non-polar environment or due to the absence of dipole relaxation processes in the tryptophan residue's surroundings.On the other hand, in proteins like melittin (at low ionic strength) the single tryptophan residue is totally exposed to the rapidly relaxing aqueous solvent [2]. Such an explanation remains in agreement with a number of observations indicating that excitation-dependent shifts of fluorescence emission can be easily detected for polar fluorophores in a polar viscous solvent [l].The fluorescence emission of most proteins is heterogeneous s...

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Fluorescence‐quenching‐resolved spectra of fluorophores in mixtures and micellar solutions

Cited by 16 publications

References 18 publications

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Dielectric relaxation in a single tryptophan protein

Red‐edge excitation fluorescence measurements of several two‐tryptophan‐containing proteins

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