Винокуров Леонид Михайлович

Кандидат химических наук

Руководитель подразделения (Группа биоинженерии репортёрных белков)

Тел.: +7 (4967) 33-05-37, +7 (4967) 73-06-53,

Эл. почта: levino@bibch.ru

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Избранные публикации

  1. Markvicheva K.N., Bilan D.S., Mishina N.M., Gorokhovatsky A.Y., Vinokurov L.M., Lukyanov S., Belousov V.V. (2011). A genetically encoded sensor for H2O2 with expanded dynamic range. Bioorg. Med. Chem. 19 (3), 1079–84 [+]

    Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by selective oxidation of redox active thiolates in proteins. Changes in intracellular [H(2)O(2)] can be tracked in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive and selective for H(2)O(2) due to the properties of its sensing domain derived from the Escherichia coli OxyR protein, many applications may benefit from an improvement of the indicator's dynamic range. We here report HyPer-2, a probe that fills this demand. Upon saturating [H(2)O(2)] exposure, HyPer-2 undergoes an up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was previously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the A233V mutation stabilizes the dimer and expands the dynamic range of the probe.

  2. Samarkina O.N., Popova A.G., Gvozdik E.Y., Chkalina A.V., Zvyagin I.V., Rylova Y.V., Rudenko N.V., Lusta K.A., Kelmanson I.V., Gorokhovatsky A.Y., Vinokurov L.M. (2009). Universal and rapid method for purification of GFP-like proteins by the ethanol extraction. Protein Expr. Purif. 65 (1), 108–13 [+]

    GFP-like fluorescent proteins (FPs) are crucial in biological and biomedical studies. The majority of FP purification techniques either include multiple time-consuming chromatography steps with a low yield of the desired product or require prior protein modification (addition of special tags). In the present work, we propose an alternative ethanol extraction-based technique previously used for GFP purification and then modified for diverse FPs originated from different sources. The following recombinant FPs were expressed using Escherichia coli M15 (pREP4) strain as a host transformed with pQE30 plasmid bearing one of the target FP genes: TagCFP, TagGFP, TagYFP, TagRFP, TurboGFP, TurboRFP, Dendra2, TurboFP602 and KillerRed. Despite their diversity, all tested recombinant FPs were successfully purified and yielded a highly homogeneous product. The method is easily scalable for purification of any amount of protein and requires no expensive reagents and equipment.

  3. Markvicheva K.N., Gorokhovatskiĭ A.I.u., Mishina N.M., Mudrik N.N., Vinokurov L.M., Lukianov S.A., Belousov V.V. (2009). Signaling function of phagocytic NADPH oxidase: activation of MAP kinase cascades in phagocytosis. Bioorg. Khim. 36 (1), 133–8 [+]

    Until recently, the production of reactive oxygen species by NADPH oxidase has been considered only in the context of the oxidative damage to pathogens inside the phagosome. However, homologues of phagocytic NADPH oxidase have been found in almost all cell types, where they produce hydrogen peroxide and thereby regulate the initial intracellular stages of MAP kinase cascades. In the present work, the activation of two MAP kinase cascades, p38 and Erk1/2, during phagocytosis has been studied. It was found that phagocytosis activates both cascades. The activation of Erkl/2 is dependent, and the activation of p38 is not dependent, on the activity of NADPH oxidase. Thus, it can be stated that the activation of MAP kinases in phagocytes during phagocytosis occurs by a mechanism similar to that operating in nonphagocytizing cells, indicating the universality of the function of NADPH oxidases in different cell types.