Fluorescence spectroscopic studies on equilibrium dipole-relaxational dynamics of Na,K-ATPase

Demchenko, Alexander P.; Apell, Hans-Jürgen; Stürmer, W.; Feddersen, Brett

doi:10.1016/0301-4622(93)85005-3

Cited by 17 publications

(5 citation statements)

References 42 publications

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“…ANS and similar molecules are essentially not fluorescent when in aqueous solution but become highly fluorescent in nonpolar solvents or when bound to proteins or membranes. These probes are highly sensitive to solvent polarity and can potentially reveal the polarity of their immediate environments 31, 35, 36…”

Section: Resultsmentioning

confidence: 99%

Stability and conformational dynamics of metallothioneins from the antarctic fish Notothenia coriiceps and mouse

et al. 2002

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The structural properties and the conformational dynamics of antarctic fish Notothenia coriiceps and mouse metallothioneins were studied by Fourier-transform infrared and fluorescence spectroscopy. Infrared data revealed that the secondary structure of the two metallothioneins is similar to that of other metallothioneins, most of which lack periodical secondary structure elements such as alpha-helices and beta-sheets. However, the infrared spectra of the N. coriiceps metallothionein indicated the presence of a band, which for its typical position in the spectrum and for its sensitivity to temperature was assigned to alpha-helices whose content resulted in 5% of the total secondary structure of the protein. The short alpha-helix found in N. coriiceps metallothionein showed an onset of denaturation at 30 degrees C and a T(m) at 48 degrees C. The data suggest that in N. coriiceps metallothionein a particular cysteine is involved in the alpha-helix and in the metal-thiolate complex. Moreover, infrared spectra revealed that both proteins investigated possess a structure largely accessible to the solvent. The time-resolved fluorescence data show that N. coriiceps metallothionein possesses a more flexible structure than mouse metallothionein. The spectroscopic data are discussed in terms of the biological function of the metallothioneins.

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Section: Resultsmentioning

confidence: 99%

Stability and conformational dynamics of metallothioneins from the antarctic fish Notothenia coriiceps and mouse

et al. 2002

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“…Consequently, the red shift of this component must be caused by relaxation of the protein matrix around the excited state. In many proteins, it is known that dipolar relaxation of the protein matrix is on the order of nanoseconds (Demchenko et al, 1993). Trp-86, which is located in a more rigid and more nonpolar environment and hence thought to be not susceptible to these relaxation processes, displays negligible shifts in the amplitudes as a function of wavelength.…”

Section: Discussionmentioning

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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“…Na,K-ATPase represents a different case, in which both dielectric relaxation and excited-state energy transfer occur on the same nanosecond time-scale. Detailed studies by combination of site-selective spectral, time-resolved and anisotropy measurements at different temperatures (146,147) allowed characterization of these processes in detail.…”

Section: Proteinsmentioning

confidence: 99%

The red‐edge effects: 30 years of exploration

2002

Self Cite

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In 1970, three laboratories independently made a discovery that, for aromatic fluorophores embedded into different rigid and highly viscous media, the spectroscopic properties do not conform to classical rules. The fluorescence spectra can depend on excitation wavelength, and the excited-state energy transfer, if present, fails at the "red" excitation edge. These red-edge effects were related to the existence of excited-state distribution of fluorophores on their interaction energy with the environment and the slow rate of dielectric relaxation of this environment. In these conditions the site-selection can be provided by variation of the energy of illuminating light quanta, and the behaviour of selected species can be followed as a function of time and other variables. These observations found extensive application in different areas of research: colloid and polymer science, molecular biophysics, photochemistry and photobiology. In particular, they led to the development of very productive methods of studying the dynamics of dielectric relaxations in protein and membranes, using the tryptophan emission and the emission of a variety of probes. These studies were extended to the time domain with the observation of new site-selective effects in emission intensity and anisotropy decays. They stimulated the emergence and development of cryogenic energy-selective and single-molecular techniques that became valuable tools in their own right in chemistry and biophysics research. Site-selection effects were discovered for electron-transfer and proton-transfer reactions if they depended on the dynamics of the environment. This review is focused on the progress in the field of red-edge effects, their applications and prospects.

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Fluorescence spectroscopic studies on equilibrium dipole-relaxational dynamics of Na,K-ATPase

Cited by 17 publications

References 42 publications

Stability and conformational dynamics of metallothioneins from the antarctic fish Notothenia coriiceps and mouse

Stability and conformational dynamics of metallothioneins from the antarctic fish Notothenia coriiceps and mouse

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

The red‐edge effects: 30 years of exploration

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