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Implication of disulfide bridge induced thermal reversibility, structural and functional stability for luciferase.

ABSTRACT: Firefly luciferase is a relatively unstable protein and commonly loses its activity at room temperature because of structural changes. The structural and functional stability of this protein is critical for its enzymatic applications. Different approaches are applied to increase the stability of this enzyme such as designing of covalent cross-links (disulfide bonds). In this study, luciferase mutants containing one or two disulfide bonds were compared to the native protein for their for their structural, thermodynamic, and functional properties. Mutant forms of P. Pyralis luciferase A²??C-A³²?C and A²??C-A³²?C/P??¹C-V???C were used. Thermodynamic and biophysical studies were carried out using UV-Vis, fluorescence, circular dichroism, luminescence spectroscopy and differential scanning calorimetry (DSC). We observed that both mutant forms of the protein were more stable than the wild-type enzyme. However, the single disulfide bond containing mutant was structurally and functionally more stable than the mutant protein containing two disulfide bonds. Furthermore, the enzymatic activity of the single disulfide bond containing mutant protein was 7-folds greater than the wild type and the double disulfide bond proteins. The A²??C-A³²?C mutation also increased the reversibility and disaggregation of the protein. The enhanced activity of the single disulfide bond mutant protein was contributed to the expansion of its active site cleft, which was confirmed by bioinformatics tools.

SUBMITTER: Naderi M

PROVIDER: S-EPMC5217160 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Project description:Developing stable nanofluids and improving their thermo-physical properties are highly important in heat transfer applications. In the present work, the stability, thermal conductivity, and rheological properties of tungsten disulphide (WS2) nanoparticles (NPs) with ethylene glycol (EG) were profoundly examined using a particle size analyzer, zeta-sizer, thermal property analyzer, rheometer, and pH measuring system. WS2 NPs were characterized by various techniques, such as XRD (X-Ray Diffraction), FTIR (Fourier Transform Infrared Spectroscopy), FESEM (Field emission scanning electron microscopy), and high-resolution transmission electron microscopy (HRTEM). The nanofluids were obtained with the two-step method by employing three volume concentrations (0.005%, 0.01%, and 0.02%) of WS2. The influence of different surfactants (Sodium dodecyl sulphate (SDS), Sodium dodecylbenzenesulfonate (SDBS), Cetyltrimethylammonium bromide (CTAB)) with various volume concentrations (0.05-2%) on the measured properties has also been evaluated. Pristine WS2/EG nanofluids exhibit low zeta potential values, i.e., -7.9 mV, -9.3 mV, and -5 mV, corresponding to 0.005%, 0.01%, and 0.02% nanofluid, respectively. However, the zeta potential surpassed the threshold (±30 mV) and the maximum values reached of -52 mV, -45 mV, and 42 mV for SDS, SDBS, and CTAB-containing nanofluids. This showed the successful adsorption of surfactants onto WS2, which was also observed through the increased agglomerate size of up to 1720 nm. Concurrently, particularly for 0.05% SDS with 0.005% WS2, thermal conductivity was enhanced by up to 4.5%, with a corresponding decrease in viscosity of up to 10.5% in a temperature range of (25-70 °C), as compared to EG. Conversely, the viscoelastic analysis has indicated considerable yield stress due to the presence of surfactants, while the pristine nanofluids exhibited enhanced fluidity over the entire tested deformation range. The shear flow behavior showed a transition from a non-Newtonian to a Newtonian fluid at a low shear rate of 10 s-1. Besides this, the temperature sweep analysis has shown a viscosity reduction in a range of temperatures (25-70 °C), with an indication of a critical temperature limit. However, owing to an anomalous reduction in the dynamic viscosity of up to 10.5% and an enhancement in the thermal conductivity of up to 6.9%, WS2/EG nanofluids could be considered as a potential candidate for heat transfer applications.

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Implication of disulfide bridge induced thermal reversibility, structural and functional stability for luciferase.

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