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26 results on '"Shubin, Vladimir V."'

12. Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin

Author

Borzova, Vera A., primary, Markossian, Kira A., additional, Chebotareva, Natalia A., additional, Kleymenov, Sergey Yu., additional, Poliansky, Nikolay B., additional, Muranov, Konstantin O., additional, Stein-Margolina, Vita A., additional, Shubin, Vladimir V., additional, Markov, Denis I., additional, and Kurganov, Boris I., additional

Published
2016
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20. Switching from blue to yellow: altering the spectral properties of a high redox potential laccase by directed evolution.

Author

Mate, Diana M., Garcia-Ruiz, Eva, Camarero, Susana, Shubin, Vladimir V., Falk, Magnus, Shleev, Sergey, Ballesteros, Antonio O., and Alcalde, Miguel

Subjects
OXIDATION-reduction reaction, LACCASE, BASIDIOMYCETES, FUNGAL enzymes, ENZYME kinetics, SACCHAROMYCES cerevisiae, PROTEIN engineering
Abstract

During directed evolution to functionally express the high redox potential laccase from the PM1 basidiomycete in Saccharomyces cerevisiae, the characteristic maximum absorption at the T1 copper site (Abs610T1Cu) was quenched, switching the typical blue colour of the enzyme to yellow. To determine the molecular basis of this colour change, we characterized the original wild-type laccase and its evolved mutant. Peptide printing and MALDI-TOF analysis confirmed the absence of contaminating protein traces that could mask the Abs610T1Cu, while conservation of the redox potential at the T1 site was demonstrated by spectroelectrochemical redox titrations. Both wild-type and evolved laccases were capable of oxidizing a broad range of substrates (ABTS, guaiacol, DMP, synapic acid) and they displayed similar catalytic efficiencies. The laccase mutant could only oxidize high redox potential dyes (Poly R-478, Reactive Black 5, Azure B) in the presence of exogenous mediators, indicating that the yellow enzyme behaves like a blue laccase. The main consequence of over-expressing the mutant laccase was the generation of a six-residue N-terminal acidic extension, which was associated with the failure of the STE13 protease in the Golgi compartment giving rise to alternative processing. Removal of the N-terminal tail had a negative effect on laccase stability, secretion and its kinetics, although the truncated mutant remained yellow. The results of CD spectra analysis suggested that polyproline helixes were formed during the directed evolution altering spectral properties. Moreover, introducing the A461T and S426N mutations in the T1 environment during the first cycles of laboratory evolution appeared to mediate the alterations to Abs610T1Cu by affecting its coordinating sphere. This laccase mutant is a valuable departure point for further protein engineering towards different fates. [ABSTRACT FROM AUTHOR]

Published
2013
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25. Glutamine synthetases of pea leaf and seed cytosol. Structure and properties

Author

Pushkin, Alexander V., primary, Antoniuk, Lyudmila P., additional, Solovieva, Nadezhda A., additional, Shubin, Vladimir V., additional, Evstigneeva, Zinaida G., additional, Kretovich, Waclaw L., additional, Cherednikova, Titiana V., additional, Tsuprun, Vladimir L., additional, Zograf, Olga N., additional, and Kiselev, Nikolai A., additional

Published
1985
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26. Effect of ionic strength and arginine on aggregation of UV-irradiated muscle glycogen phosphorylase b.

Author

Eronina, Tatiana B., Mikhaylova, Valeriya V., Chebotareva, Natalia A., Shubin, Vladimir V., and Kurganov, Boris I.

Subjects
*ARGININE, *IONIC strength, *GLYCOGEN phosphorylase, *CLUSTERING of particles, *ULTRAVIOLET radiation
Abstract

In this work the effect of ionic strength and arginine on the kinetics of aggregation of UV-irradiated muscle glycogen phosphorylase b (UV-Ph b ) was studied using dynamic light scattering at 37 °C at various ionic strengths (0.02–0.7 M). Under these conditions the rate-limiting stage of the overall aggregation process is the structural reorganization of UV-Ph b , which can be characterized by the first order rate constant k I . It was shown that an increase in NaCl concentration caused a decrease in the k I value, suggesting a slowdown of the UV-Ph b structural reorganization. Circular dichroism data confirmed this conclusion. Arginine is widely used in biotechnology as an agent suppressing protein aggregation. However, arginine is a charged molecule, and, when studying the action of arginine on protein aggregation, the effects of ionic strength should be taken into account. To evaluate the effect of arginine, experiments were conducted at fixed values of ionic strength (0.15 M and 0.5 M). It was shown that at a low ionic strength arginine (0–0.13 M) accelerated the process of protein aggregation, whereas at higher ionic strength arginine (0–0.48 M) acted as an aggregation suppressor. [ABSTRACT FROM AUTHOR]

Published
2018
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