Recent Developments in Frequency-Domain Fluorometry

Lakowicz, Joseph R.; Maliwal, Badri P.; Gratton, Enrico

doi:10.1080/10739148508543578

Cited by 21 publications

(3 citation statements)

References 44 publications

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“…Fluorescence Lifetime Measurements. Fluorescence lifetimes were measured using multifrequency phase fluorometry (Lakowicz et al, 1985). The measured phase shifts, , at a modulation frequency, , of the exciting light are related to the fluorescence decay in the time domain…”

Section: Methodsmentioning

confidence: 99%

Fluorescence study of the three tryptophan residues of the pore-forming domain of colicin A Using multifrequency phase fluorometry

Vos¹,

Engelborghs

Izard³

et al. 1995

Biochemistry

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We have identified the steady-state and time-resolved fluorescence of the three tryptophan residues (Trp-86, Trp-130, and Trp-140) of the pore-forming domain of colicin A using site-directed mutagenesis in order to construct two- and one-tryptophan-containing mutant proteins. Fluorescence lifetimes were measured via multifrequency phase fluorometry. The fluorescence of the pore-forming domain of colicin A is dominated by Trp-140 which contributes almost 53% to the fluorescence intensity. Mutation of Trp-140 results in a decrease in fluorescence quantum yield and average lifetime. Colicin A wild-type and all mutant proteins display multiple lifetimes which belong to three different lifetime classes: 0.38-0.57 ns for tau 1, 1.6-1.87 ns for tau 2, and 3.6-4.41 ns for tau 3 at pH 5. At pH 7, the three classes are 0.64-0.89 ns for tau 1, 2.01-2.19 ns for tau 2, and 4.23-4.94 ns for tau 3. This pH effect influences all the lifetimes and must be attributed to a general conformational change. In wild-type colicin A, tau 3 originates mainly from Trp-140 while Trp-86 and Trp-130 both provide a major contribution to tau 2. The pH dependence of the fluorescence intensity gives rise to a pKa of 5.2. The different lifetime components of two of the three single-tryptophan-containing mutants show different quenching properties toward acrylamide, indicating that each lifetime is coupled to a different microenvironment. The linear combination of the lifetimes of the single tryptophans into pairs simulates very well the behavior of the two-tryptophan-containing mutants except for one, the mutant containing Trp-86 and Trp-130. The lifetimes of the wild-type protein can only be obtained by the linear combination of the lifetimes from the mutant containing the tryptophan pair Trp-86/Trp-130 and the mutant containing Trp-140. Mutual energy transfer between Trp-86 and Trp-130 is assumed to be the explanation of this deviation since the mutant proteins display no structural or dynamic aberrances. The calculated energy transfer efficiency amounts to 65% for energy transfer from Trp-86 to Trp-130 and 21% for the reverse transfer and is in agreement with our measurements.

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Section: Methodsmentioning

confidence: 99%

Fluorescence study of the three tryptophan residues of the pore-forming domain of colicin A Using multifrequency phase fluorometry

Vos¹,

Engelborghs

Izard³

et al. 1995

Biochemistry

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“…More recently, preliminary reports (Yoshida el al., 1986;Garland & Birmingham, 1986) describe the implementation of polarized fluorescence depletion methods in the frequency domain. Following the lead of recent technical advances in polarized phase fluorometry (Lakowicz et al, 1985), rotational correlation times can be deduced from the amplitudes and phases of fluorescence depletion signal frequencies measured by lock-in amplifiers phase-locked to the intensity and polarization modulation of the incident illumination.…”

Section: Underlving Principlesmentioning

confidence: 99%

Fluorescence photobleaching recovery techniques for translational and slow rotational diffusion in solution and on cell surfaces

Koppel

1986

Biochemical Society Transactions

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“…The biosciences literature is rich in both experimental and theoretical contributions which serve as a basis for the application of fluorescence measurements in polymer science. 12,[26][27][28][29][30][31][32] Time resolved fluorescence anisotropy, phase modulated fluorescence, continuous wave fluorescence anisotropy, and continuous wave fluorescence spectroscopy are four methods used routinely in fluorescence work. The techniques yield both static and dynamic information about the molecular state and, in the case of fluorescence anisotropy, knowledge about molecular orientation is obtained.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence anisotropy sensor and its application to polymer processing and characterization

Bur

Roth

Thomas

2000

Review of Scientific Instruments

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An optical sensor containing polarizing optical components has been constructed to monitor fluorescence anisotropy during polymer processing and to carry out remote sensing of polymer products doped with fluorescent dyes. The sensor is a compact unit that is used to polarize incident excitation light as well as to analyze the polarization of generated fluorescent light. Optical fibers are used to carry light between the sensor head and the light source and detecting equipment. The anisotropy measurement yields information about the orientation of a fluorescent dye molecule that has been doped into polymer matrix. Fluorescent dyes that have geometrical asymmetry in their molecular structure are used. Experiments are described for which the sensor is positioned in line during extrusion, during specimen extension, and where the sensor is used to carry out area scans of films and sheets. Measurements were made on polyethylene, polyethylene terephthalate, and polybutadiene resins that contained a low concentration of fluorescent dye. ͓S0034-6748͑00͒04103-4͔

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Recent Developments in Frequency-Domain Fluorometry

Cited by 21 publications

References 44 publications

Fluorescence study of the three tryptophan residues of the pore-forming domain of colicin A Using multifrequency phase fluorometry

Fluorescence study of the three tryptophan residues of the pore-forming domain of colicin A Using multifrequency phase fluorometry

Fluorescence photobleaching recovery techniques for translational and slow rotational diffusion in solution and on cell surfaces

Fluorescence anisotropy sensor and its application to polymer processing and characterization

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