Fluorescence Quenching: Theory and Applications

Eftink, Maurice R.

doi:10.1007/0-306-47058-6_2

Cited by 131 publications

(126 citation statements)

References 227 publications

(243 reference statements)

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“…To examine the accessibility and relative location of melittin in various types of micelles, fluorescence quenching experiments were performed with acrylamide, which is a widely used neutral aqueous quencher of tryptophan fluorescence (Eftink 1991b). The results of these experiments for quenching of the tryptophan residue of melittin by acrylamide in various micellar systems are plotted in Fig.…”

Section: Acrylamide Quenching Of Melittin Tryptophan Fluorescencementioning

confidence: 99%

Effect of micellar charge on the conformation and dynamics of melittin

Raghuraman

Chattopadhyay

2004

Eur Biophys J

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Electrostatic interactions play a crucial role in modulating and stabilizing molecular interactions in membranes and membrane-mimetic systems such as micelles. We have monitored the change in the conformation and dynamics of the cationic hemolytic peptide melittin bound to micelles of various charge types, utilizing fluorescence and circular dichroism (CD) spectroscopy. The sole tryptophan of melittin displays a red-edge excitation shift (REES) of 3-6 nm when bound to anionic, nonionic, and zwitterionic micelles. This suggests that melittin is localized in a restricted environment, probably in the interfacial region of the micelles, and this region offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state tryptophan in melittin. Further, the rotational mobility of melittin is considerably reduced in these micelles and is found to be dependent on the surface charge of micelles. Interestingly, our results show that melittin does not partition into cetyltrimethylammonium bromide (CTAB) micelles owing to electrostatic repulsion between melittin and CTAB micelles, both of which carry a positive charge. In addition, the fluorescence lifetime of melittin is modulated in micelles of different charge types. The lowest mean fluorescence lifetime is observed in the case of melittin bound to anionic sodium dodecyl sulfate (SDS) micelles. CD spectroscopy shows that micelles induce significant helicity to melittin, with maximum helicity being induced in the case of melittin bound to SDS micelles. Fluorescence quenching measurements using the neutral aqueous quencher acrylamide show differential accessibility of melittin in various types of micelles. Taken together, our results show that micellar surface charge can modulate the conformation and dynamics of melittin. These results could be relevant to understanding the role of the surface charge of membranes in the interaction of membrane-active, amphiphilic peptides with membranes.

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Section: Acrylamide Quenching Of Melittin Tryptophan Fluorescencementioning

confidence: 99%

Effect of micellar charge on the conformation and dynamics of melittin

Raghuraman

Chattopadhyay

2004

Eur Biophys J

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“…Specifically, iodide ion and acrylamide were used to probe the environment of Trp 21. Iodide has been used extensively to quench tryptophans that are in polar regions of proteins, particularly near positively charged sites (Stayton 8z Sligar, 1990;Eftink, 1991). Trp 21 of rat Al-1 GST is relatively insensitive to iodide quenching.…”

Section: Fluorescence Quenchingmentioning

confidence: 99%

“…The data suggest that the difference in quenching constants for the individual lifetime components, Ksv,, or their fractional intensities, f,, is more pronounced in the the presence of S-hexyl GSH than in its absence. Assuming that groundstate heterogeneity provides the observed wavelength dependence of the apparent Ksv values (Eftink, 1991), the data shown in these modified Stern-Volmer plots have also been analyzed by nonlinear regression, using the equation F/Fo = EA/( 1 + KsviQ). Unique solutions of this analysis were obtained for the data at the extreme wavelengths, 320 nm and 380 nm.…”

Section: Fluorescence Quenchingmentioning

confidence: 99%

“…For iodide quenching experiments, samples with varying concentrations of iodide were kept at constant ionic strength by addition of an appropriate concentration of KCl. The Stern-Volmer quenching constants for the individual lifetime components were obtained from the relationship F/Fo = Cf,/( 1 + [ Q ] Ksu;), where Fo and Fare the wavelength-dependent fluorescence intensities in the absence and presence of quencher, respectively, Q is the concentration of quencher, f, is the fractional intensity of the ith component, and Ksu; is the Stern-Volmer quenching constant of the ith component (Eftink [1991] and references therein). Nonlinear regression utilizing this equation was accomplished with the software package Enzfitter@.…”

Section: Steady-state Fluorescencementioning

confidence: 99%

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Fluorescence characterization of Trp 21 in rat glutathione S‐transferase 1–1: Microconformational changes induced by S‐hexyl glutathione

et al. 1993

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The glutathione S-transferase (GST) isoenzyme Al-1 from rat contains a single tryptophan, Trp 21, which is expected to lie within a-helix 1 based on comparison with the X-ray crystal structures of the pi-and mu-class enzymes. Steady-state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand-induced conformational changes in this region of the protein. Addition of S-hexyl glutathione to GST isoenzyme Al-1 causes an increase in the steady-state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequencydomain excited-state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substratefree GST. In the presence of S-hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate-free protein, the short lifetime component contributes 9-16070 of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S-hexyl glutathione. Steady-state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide-quenching experiments at several emission wavelengths indicate that the long-wavelength components become quenched more easily in the presence of S-hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the "H-site" is occupied.

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“…Acrylamide is a fluorescence quencher known to reduce AP fluorescence via bimolecular collisions (27). The slope of the graph derived from acrylamide quenching experiments provides the Stern-Volmer constant (K sv ), which reflects the sensitivity, exposure, and accessibility of AP toward the quencher.…”

Section: Acrylamide Quenching and Temperature-dependent Fluorescencementioning

confidence: 99%

Fluorescence Probing of Aminofluorene-Induced Conformational Heterogeneity in DNA Duplexes

Jain

Reshetnyak²,

Gao³

et al. 2008

Chem. Res. Toxicol.

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Fluorescence spectroscopy was used to study carcinogen-induced conformational heterogeneity in DNA duplexes. The fluorophore 2-aminopurine (AP) was incorporated adjacent (5′) to the lesion (G*) in eight different DNA duplexes [d(5′-CTTCTPG*NCCTC-3′):d(5′-GAGGNXTAGAAG-3′), G* = FAF adduct, P = AP, N = G, A, C, T, and X = C, A] modified by FAF [N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene], a fluorine-tagged model DNA adduct derived from the potent carcinogen 2-aminofluorene. Steady-state measurements showed that fluorescence intensity and Stern-Volmer constants (K sv ) derived from acrylamide quenching experiments decreased for all carcinogen-modified duplexes relative to the controls, which suggests greater AP stacking in the duplex upon adduct formation. Conformation-specific stacking of AP with the neighboring adduct was evidenced by a sequence-dependent variation in fluorescence intensity, position of emission maximum, degree of emission quenching by acrylamide, and temperature-dependent spectral changes. The magnitude of stacking was in the order of FAF residue in base-displaced stacked (S) > minor groove wedged (W) > major groove B type (B). This work represents a novel utility of AP in probing adduct-induced conformational heterogeneities in DNA duplexes.

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Fluorescence Quenching: Theory and Applications

Cited by 131 publications

References 227 publications

Effect of micellar charge on the conformation and dynamics of melittin

Effect of micellar charge on the conformation and dynamics of melittin

Fluorescence characterization of Trp 21 in rat glutathione S‐transferase 1–1: Microconformational changes induced by S‐hexyl glutathione

Fluorescence Probing of Aminofluorene-Induced Conformational Heterogeneity in DNA Duplexes

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