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  • 1
    Electronic Resource
    Electronic Resource
    [S.l.] : American Institute of Physics (AIP)
    Review of Scientific Instruments 65 (1994), S. 1485-1495 
    ISSN: 1089-7623
    Source: AIP Digital Archive
    Topics: Physics , Electrical Engineering, Measurement and Control Technology
    Notes: Significant improvements in the performances of the Super-ACO storage ring free-electron laser (FEL) at 800 MeV have been obtained recently: enhancement of the output power in the ultraviolet, laser duration of 10 h for the same injection of positrons, long-term stability with a micropulse of 60 ps FWHM. A first series of experiments using this FEL has then been successfully performed. Taking advantage of the time structure, the polarization and the high power of the source at 350 nm, the polarized fluorescence decays of the reduced nicotinamide adenine dinucleotide coenzyme (NADH) were studied in aqueous solution, using the single-photon counting (SPC) technique. The experimental setup is described, including the Super-ACO FEL characteristics and diagnostics. The FEL working point has been first optimized by monitoring the SPC apparatus function. A complete fluorescence experiment required about 30 min of data acquisition, during which the best integrated instrumental response had a FWHM of 110 ps. Measurements performed in such a way lead to the unambiguous separation of two close lifetime components of 0.28 and 0.62 ns in the fluorescence decays of NADH at 20 °C, in good agreement with previous works. The thermodynamic parameters obtained from temperature studies show that the NADH fluorescence heterogeneity is consistent with the ground-state folding equilibrium of the coenzyme, as characterized by many other spectroscopic techniques. From the fluorescence anisotropy decays, an apparent hydrodynamic radius of about 6 A(ring) is determined, while on the other hand, a large initial depolarization of the fluorescence indicates a fast independent motion of the nicotinamide ring. The quality of the collected data fully meets the requirements for the study of more complex systems such as fluorescent compounds bound to proteins or membranes. Thus, the feasibility of use of a storage ring UV FEL for this type of time-resolved experiments on the subnanosecond time scale has been demonstrated.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Analytical chemistry 65 (1993), S. 1028-1034 
    ISSN: 1520-6882
    Source: ACS Legacy Archives
    Topics: Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Biochemistry 33 (1994), S. 2610-2619 
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Copenhagen : International Union of Crystallography (IUCr)
    Acta crystallographica 50 (1994), S. 398-403 
    ISSN: 1399-0047
    Source: Crystallography Journals Online : IUCR Backfile Archive 1948-2001
    Topics: Chemistry and Pharmacology , Geosciences , Physics
    Notes: Early steps in the crystallization process of pancreatic ribonuclease have been investigated by time-dependent fluorescence anisotropy, using a labeled protein as a fluorescent probe. Previous experiments have shown that steady-state fluorescence anisotropy is sensitive to protein–protein interactions and can be used to find new crystallization conditions. The present work describes an attempt, by means of time-resolved experiments, to detect and characterize species appearing in the early stages of the crystallization pathway. Fluorescence anisotropy decay was measured with synchrotron radiation as a light source under a variety of conditions where it is known that the solutions tend towards crystallization; the decay was analyzed by a maximum-entropy method that calculates a rotational correlation-time distribution. Fluorescence anisotropy originates in the Brownian rotatory motion of macromolecules and the values of the correlation times are related to the size and shape of different species present in the solution. In the presence of high salt concentrations, a bimodal distribution is always observed. Whereas a peak of protein monomer is still present, a second peak appears as a stable intermediate in the crystallization pathway. The correlation time of this new species varies between two and three times the correlation time of the monomer. The second peak is possibly the symmetrical dimer of the ribonuclease molecules commonly observed in all the high-salt crystal forms.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Nuclear Instruments and Methods in Physics Research Section A: 341 (1994), S. 132-137 
    ISSN: 0168-9002
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Physics
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-1017
    Keywords: Fluorescence quenching ; protein conformation ; ribonuclease A
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract The fluorescence properties of ribonuclease labelled at its active site with N-(iodoacetylamino)-ethyl-5-naphthylamine-1-sulfonic acid have been studied at different temperatures and in the presence of acrylamide. The rate constant for the quenching of the fluorescence of labelled ribonuclease by acrylamide is apparently not limited by the “accessibility” of the probe: similar values are obtained for the native and denatured states of the protein. Instead, acrylamide seems to be a rather inefficient quencher of this fluorescent group ((acetamidoamino) ethyl-5-naphtylamine-1-sulfonic acid), as shown by non-linear Stern-Volmer representations, biphasic decay kinetics, and a low value of the rate constant. The fluorescence intensity of the native state of the labelled protein is highly sensitive to temperature and exhibits a 20% decrease for an increase of temperature of from 10°C to 30°C, independent of solvent viscosity. This thermal quenching is specific for the native conformation and disappears when the protein is unfolded. When the fluorescence life-time of the label is shortened by addition of acrylamide, the effect of temperature becomes identical for native and unfolded structures. This suggests that the cause of the thermal quenching is the presence of conformational fluctuations within the native protein which apparently take place in the time range from 35 to 200 ns.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    European biophysics journal 13 (1986), S. 291-299 
    ISSN: 1432-1017
    Keywords: Hexokinase (wheat germ) ; conformational change, hysteresis ; pulse fluorimetry ; synchrotron radiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract The conformational properties of wheat germ hexokinase LI, a monomeric enzyme showing non-Michaelian kinetics, have been studied by polarised pulse fluorimetry using synchrotron radiation as an excitation light source. The fluorescence decays and the fluorescence anisotropy decays of tryptophyl residues were measured with excitation at 300 nm. At pH 8.5, we found that the “mnemonical” temperature-dependent transition did not induce any detectable structural change in the protein. This rules out modifications of the aggregation state of hexokinase during the transition as well as important conformational changes in the tertiary structure. At pH 6.1, a temperature-dependent transition of the enzyme-glucose binary complex is observed: rapid, large amplitude, internal motions appear in the structure when the temperature is raised from-1°C to 30°C. Full standard activity is retained during this dynamic change. In the experiments described here we obtained an active fluorescent derivative by reacting hexokinase with N-(iodoacetylaminoethyl)-5-naphtylamine-1-sulfonic acid (1,5-IAEDANS), in the presence of glucose. Polarised fluorescence decay measurements indicate that the label is exposed to the solvent and very mobile, which makes it ineffective as a probe for the conformational properties of hexokinase.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1573-4994
    Keywords: Lifetime distributions ; heterogeneities ; associated dynamics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Abstract After purification to homogeneity by Bio-Rex 70 ion exchange chromatography, micromolar solutions ofNaja nigricollis cardiotoxin were found to contain significant amounts of aggregates, as detected by time-resolved polarized fluorescence of its single tryptophan residue. The level of cardiotoxin aggregation depends strongly and reversibly on the protein concentration and pH. However, supplementary reverse-phase HPLC completely suppresses this aggregation, resulting in all cases in fluorescence anisotropy decays characteristic of the pure cardiotoxin monomer. The self-association properties of cardiotoxin, in the presence of a possible cofactor eliminated by the HPLC step, may be functionally relevant, and would deserve further investigation. The physical heterogeneity of the cardiotoxin samples required an appropriate model for the analysis of fluorescence depolarization, which was iteratively improved by comparison with experimental results. In this way, an approximate molar fraction of 10–15% aggregated cardiotoxin at a 90ΜM total protein concentration, pH 7, was determined. The fluorescence of the partly aggregated samples is significantly perturbed as compared to the HPLC-treated monomer, indicating that the cardiotoxin aggregate must have an increased average fluorescence lifetime and a strongly decreased initial anisotropy. The decrease in initial anisotropy suggests either an increased mobility of the tryptophan residue upon aggregation or fast energy transfers between residues of different cardiotoxin molecules brought within a short distance in the aggregate. This study illustrates the high sensitivity of the time-resolved fluorescence technique, through both total fluorescence and anisotropy parameters, to low levels of physical or chemical heterogeneity in a protein sample.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1573-4994
    Keywords: Two-photon excitation ; fluorescence correlation microscopy ; translational diffusion ; single-molecule detection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Abstract We report on the application of fluorescence correlation microscopy under two-photon excitation of fluorophores of biological interest: FITC–dextran (MW, from 20 to 150 kDa), green fluorescent protein (MW, 27 kDa), and fluorescein (MW, 330 Da). Under these experimental conditions, the translational diffusion coefficients of these molecules in aqueous solutions derived from the fluorescence intensity autocorrelation function were determined for the first time and were found to be 24 × 10−7, 8.2 × 10−7, and 3 × 10−7 cm2 s−1 for 150-kDa FITC–dextran, green fluorescent protein, and fluorescein, respectively. These results are discussed in connection with previously reported results obtained by different methods. The great sensibility of the system has been applied to single-molecule detection of the smaller fluorophore, fluorescein.
    Type of Medium: Electronic Resource
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