Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry

Dalbey, Ross E.; Weiel, Jurgen; Perkins, William J.; Yount, Ralph G.

doi:10.1016/0165-022x(84)90030-7

Cited by 11 publications

(4 citation statements)

References 24 publications

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“…The FRET efficiency (E) depends on the distance (R) separating the dipole moments of the donor and acceptor probes according to the equation: where R 0 is the Fo ¨rster critical distance (the distance at which the efficiency of energy transfer is 50%). For the 1,5-IAEDANS-DDPM probe pair, we used the R 0 value of 29 Å published by Dalbey et al (1984) and the value of 42.8 Å calculated by Kasprzak for the 1,5-IAEDANS-DABMI probe pair (Kasprzak et al, 1988).…”

Section: Methodsmentioning

confidence: 99%

A Conformational Change in F-Actin When Myosin Binds: Fluorescence Resonance Energy Transfer Detects an Increase in the Radial Coordinate of Cys-374

Moens

1997

Biochemistry

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Interactions of myosin with actin filaments probably induce conformational changes in actin which are crucial for its function. Fluorescence resonance energy transfer spectroscopy can determine changes in distance (range 10-100 A) between two probes and therefore can sense conformational changes in proteins. We have investigated changes in the radial coordinates of fluorescent probes bound to Cys-374 of F-actin when either of the isozymes (S1A1 and S1A2) of myosin subfragment 1 (S-1) bind. Using 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide as donor and acceptor probes, respectively, we calculated a radius of 13-14 A. This distance increased by approximately 4.5 A upon addition of S-1. No differences were detected between the effects of S1A1 and S1A2. This increase is reversed by MgATP. The average position of the probes on Cys-374 is closer to the filament axis than expected from the current models of F-actin. S-1 increases the radial position of Cys-374 either by direct interaction or via an allosteric conformational change associated with its binding.

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Section: Methodsmentioning

confidence: 99%

A Conformational Change in F-Actin When Myosin Binds: Fluorescence Resonance Energy Transfer Detects an Increase in the Radial Coordinate of Cys-374

Moens

1997

Biochemistry

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“…Weber devised an analytical solution to obtain the decay times of N components from data measured at N frequencies (35,36). This method appears to be unstable (37,38) and sensitive to the small systematic errors which were present in data obtained from the fixed-frequency instruments (38)(39)(40). This method has not been used to recover the emission spectra of the components.…”

Section: Introductionmentioning

confidence: 99%

“…There is no general method to recover the component decay times (t¡(X)) and amplitudes ( ;( )) from these limited data ( *8 ( ) and Tmapp(X)). These apparent values have been used occasionally to fit the data in terms of the multiexponential model (40,55). While this approach, when used with caution, may yield acceptable results, the fitting to apparent lifetimes should be discouraged.…”

Section: Introductionmentioning

confidence: 99%

Resolution of multicomponent fluorescence emission using frequency-dependent phase angle and modulation spectra

Lakowicz¹,

Jayaweera²,

Szmacinski³

et al. 1990

Anal. Chem.

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We describe a new fluorescence method that allows the resolution of both the decay times and emission spectra of mixtures of fluorophores. This method is completely general and does not require any assumptions or knowledge of the decay times or emission spectra of the individual fluorophores. We use the phase angle spectra and modulation spectra of the mixture, measured over a range of suitable light modulation frequencies and emission wavelengths. These data are analyzed by nonlinear least-squares analysis to recover the emission spectra and the associated decay times. The principle of the method and the nature of the data are illustrated by using two-component mixtures with increasing spectral overlap. We then demonstrate the recovery of minor components, of structure emission spectra, and of a three-component mixture with completed overlapping emission spectra. And finally, we describe the resolution of a two-component mixture with decay times of 0.8 and 1.4 ns using modulation frequencies up to 774 MHz.

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“…Regardless of whether the fluorescence is being used for analytical purposes, such as determining the amount of bound species in an immunoassay, or as a probe of a macromolecule, it is often necessary to determine the parameters that describe the heterogeneous emission, these being the lifetimes and fractional intensities in the decay law. Both the time-domain (4-7) and phasemodulation (7)(8)(9)(10)(11)(12)(13) techniques for the measurement of these decays have improved significantly in recent years. In particular, the recent appearance of variable frequency phase fluorometers (14,15) permit measurements up to 2 GHz (16) and allow resolution of complex lifetimes and anisotropy decays even on the picosecond time scale (17).…”

mentioning

confidence: 99%

Resolution of multicomponent fluorescence emission by phase sensitive detection at multiple modulation frequencies

Keating-Nakamoto¹,

Cherek²,

Lakowicz³

1987

Anal. Chem.

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Phase sensitive emission spectra recorded at multiple frequencies were used to determine the lifetimes and steady-state spectra of the fluorophores In two-component mixtures. This analysis does not require any previous knowledge of the lifetimes, fractional Intensities, or the emission spectra and is thus an Improvement over single frequency phase sensitive detection which requires known emission spectra or known lifetimes. Phase sensitive emission spectra are recorded at several frequencies and arbitrarily chosen detector phase angles. The data are analyzed by use of nonlinear least squares to recover the lifetimes and wavelength-dependent fractional Intensities. The latter values determine the emission spectrum and relative intensity of each component in the mixture. Using this technique, we resolved two-component mixtures of fluorescein and 9amlnoacrldlne, 2-(p-toluidinyl)-naphthalene-6-sulfonlc acid and 6-proplonyl-2-(dlmethylamlno)naphthalene, and N-acetyl-L-tyrosinamide and N-acetyl-L-tryptophanamide. In these mixtures the lifetimes differ by about 2-fold. Analysis of simulated data is presented to Illustrate the requirements for a satisfactory resolution. For simulated two-component mixtures, the components can be resolved If the lifetimes differ by 2-fold or greater, even with extensive overlap of the emission spectra. Registry No.

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Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry

Cited by 11 publications

References 24 publications

A Conformational Change in F-Actin When Myosin Binds: Fluorescence Resonance Energy Transfer Detects an Increase in the Radial Coordinate of Cys-374

A Conformational Change in F-Actin When Myosin Binds: Fluorescence Resonance Energy Transfer Detects an Increase in the Radial Coordinate of Cys-374

Resolution of multicomponent fluorescence emission using frequency-dependent phase angle and modulation spectra

Resolution of multicomponent fluorescence emission by phase sensitive detection at multiple modulation frequencies

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