Frequency-Domain Fluorescence Spectroscopy

Lakowicz, Joseph R.; Gryczyński, Ignacy

doi:10.1007/0-306-47057-8_5

Cited by 201 publications

(309 citation statements)

References 74 publications

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“…As shown by fluorescence measurements (Fig. 3), following the re-organization of the secondary structure, Trp residues are more solvent-exposed in native apo-OASS (maximum at 343 nm) than in holo-OASS (maximum at 338 nm) (43). In holo-OASS, phosphorescence studies (18) indicate that Trp-161 is embedded in a more polar environment than Trp-50.…”

Section: Discussionmentioning

confidence: 81%

Role of Pyridoxal 5′-Phosphate in the Structural Stabilization of O-Acetylserine Sulfhydrylase

Bettati¹,

Benci²,

Campanini³

et al. 2000

Journal of Biological Chemistry

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Proteins belonging to the superfamily of pyridoxal 5-phosphate-dependent enzymes are currently classified into three functional groups and five distinct structural fold types. The variation within this enzyme group creates an ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions. The number of known three-dimensional structures of pyridoxal 5-phosphate-dependent enzymes is rapidly increasing, but only for relatively few have the folding mechanisms been characterized in detail. The dimeric O-acetylserine sulfhydrylase from Salmonella typhimurium belongs to the ␤-family and fold type II group. Here we report the guanidine hydrochloride-induced unfolding of the apo-and holoprotein, investigated using a variety of spectroscopic techniques. Data from absorption, fluorescence, circular dichroism, 31 P nuclear magnetic resonance, time-resolved fluorescence anisotropy, and photon correlation spectroscopy indicate that the O-acetylserine sulfhydrylase undergoes extensive disruption of native secondary and tertiary structure before monomerization. Also, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater conformational stability than the apoenzyme form. The data are discussed in light of the fact that the role of the coenzyme in structural stabilization varies among the pyridoxal 5-phosphate-dependent enzymes and does not seem to be linked to the particular enzyme fold type.

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Section: Discussionmentioning

confidence: 81%

Role of Pyridoxal 5′-Phosphate in the Structural Stabilization of O-Acetylserine Sulfhydrylase

Bettati¹,

Benci²,

Campanini³

et al. 2000

Journal of Biological Chemistry

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“…However, Trp fluorescence is complex because of different rotamers in the ground state and the two nearly degenerate electronic states ( 1 L a , 1 L b ) with perpendicular transition moments. Accordingly, numerous studies (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) have focused on the lifetime, quantum yield, Stokes shift, and fluorescence anisotropy. Most of these studies were made with picosecond or nanosecond time resolution (3,(6)(7)(8)(9)(10).…”

mentioning

confidence: 99%

Femtosecond dynamics of rubredoxin: Tryptophan solvation and resonance energy transfer in the protein

Zhong

Pal

Zhang

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

138

134

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We report here studies of tryptophan (Trp) solvation dynamics in water and in the Pyrococcus furiosus rubredoxin protein, including the native and its apo and denatured forms. We also report results on energy transfer from Trp to the iron-sulfur [Fe-S] cluster. Trp fluorescence decay with the onset of solvation dynamics of the chromophore in water was observed with femtosecond resolution (Ϸ160 fs; 65% component), but the emission extended to the picosecond range (1.1 ps; 35% component). In contrast, the decay is much slower in the native rubredoxin; the Trp fluorescence decay extends to 10 ps and longer, reflecting the local rigidity imposed by residues and by the surface water layer. The dynamics of resonance energy transfer from the two Trps to the [Fe-S] cluster in the protein was observed to follow a temporal behavior characterized by a single exponential (15-20 ps) decay. This unusual observation in a protein indicates that the resonance transfer is to an acceptor of a well-defined orientation and separation. From studies of the mutant protein, we show that the two Trp residues have similar energy-transfer rates. The critical distance for transfer (R 0) was determined, by using the known x-ray data, to be 19.5 Å for Trp-36 and 25.2 Å for Trp-3, respectively. The orientation factor ( 2 ) was deduced to be 0.13 for Trp-36, clearly indicating that molecular orientation of chromophores in the protein cannot be isotropic with 2 value of 2͞3. These studies of solvation and energy-transfer dynamics, and of the rotational anisotropy, of the wild-type protein, the (W3Y, I23V, L32I) mutant, and the fmetPfRd variant at various pH values reveal a dynamically rigid protein structure, which is probably related to the hyperthermophilicity of the protein.T ryptophan (Trp) is the most important fluorophore among amino acid residues for optical probing of proteins. However, Trp fluorescence is complex because of different rotamers in the ground state and the two nearly degenerate electronic states ( 1 L a , 1 L b ) with perpendicular transition moments. Accordingly, numerous studies (1-10) have focused on the lifetime, quantum yield, Stokes shift, and fluorescence anisotropy. Most of these studies were made with picosecond or nanosecond time resolution (3, 6-10). To probe the local protein dynamics, Trp solvation by neighboring soft͞rigid water molecules, or by other polar amino acid residues, must be resolved on the femtosecond time scale. Moreover, such studies are important for examining the nature of resonance energy transfer (RET) that is used for deducing distances and orientations between the Trp and quenchers in the protein (e.g., see refs. 3 and 10, and references therein).We choose the hyperthermophilic iron-sulfur protein, Pyrococcus furiosus rubredoxin (PfRd), as a prototype system (Fig. 1). The high-resolution x-ray crystallographic structures of PfRd at 0.95 Å and its formylmethionine variant (fmetPfRd) at 1.2 Å, have been recently reported (11). PfRd is a small protein of 53 amino acid residues with an iron a...

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“…The intensity decays were recovered from the FD data in terms of a multiexponential model using nonlinear least squares analysis Lakowicz et al, 1984;Lakowicz and Gryczinski, 1991;Kang and Lakowicz, 2001;Kang and Kostov, 2002;Kang et al, 2002a,b,c): (2) where the preexponential factor α i is the amplitude of each component, Σα i = 1.0, τ i is the decay time, and n is the number of exponential components. Mean lifetimes were calculated by: (3) where f i is the fractional steady-state contribution of each component to the total emission, and Σf i is normalized to unity.…”

Section: Fd Intensity and Anisotropy Decay Measurementsmentioning

confidence: 99%

“…δφ and δm are the experimental uncertainties in the measured phase and modulation values and were set at 0.2° and 0.005, respectively. The FD anisotropy decays were also analyzed in terms of the multiexponential model using nonlinear least squares analysis (Lakowicz and Gryczinski, 1991;Lakowicz et al, 1993;Kang et al, 2002a,b,c): (10) where g i is the amplitude of the anisotropy component with a rotational correlation time θ i , Σg i = 1.0, and r 0 is the anisotropy in the absence of rotational diffusion. The total anisotropy r 0 was a fitted parameter.…”

Section: Fd Intensity and Anisotropy Decay Measurementsmentioning

confidence: 99%

Dynamics of Supercoiled and Linear pBluescript II SK(+) Phagemids Probed with a Long-lifetime Metal-ligand Complex

Kang¹,

Son²,

Choi³

et al. 2005

BMB Reports

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We extended the measurable time scale of DNA dynamics to microsecond using [Ru(phen) 2 (dppz)] 2+ (phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine) (RuPD), which displays a mean lifetime near 500 ns. To evaluate the usefulness of this luminophore (RuPD) for probing nucleic acid dynamics, its intensity and anisotropy decays when intercalated into supercoiled and linear pBluescript (pBS) II SK(+) phagemids were examined using frequency-domain fluorometry with a blue lightemitting diode (LED) as the modulated light source. The mean lifetime for the supercoiled phagemids (< τ > = 489.7 ns) was somewhat shorter than that for the linear phagemids (< τ > = 506.4 ns), suggesting a more efficient shielding from water by the linear phagemids. The anisotropy decay data also showed somewhat shorter slow rotational correlation times for supercoiled phagemids (997.2 ns) than for the linear phagemids (1175.6 ns). The slow and fast rotational correlation times appear to be consistent with the bending and torsional motions of the phagemids, respectively. These results indicate that RuPD can have applications in studies of both bending and torsional dynamics of nucleic acids.

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Frequency-Domain Fluorescence Spectroscopy

Cited by 201 publications

References 74 publications

Role of Pyridoxal 5′-Phosphate in the Structural Stabilization of O-Acetylserine Sulfhydrylase

Role of Pyridoxal 5′-Phosphate in the Structural Stabilization of O-Acetylserine Sulfhydrylase

Femtosecond dynamics of rubredoxin: Tryptophan solvation and resonance energy transfer in the protein

Dynamics of Supercoiled and Linear pBluescript II SK(+) Phagemids Probed with a Long-lifetime Metal-ligand Complex

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