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Mishin A.V.,Moscow Institute of Physics and Technology | Luginina A.P.,Moscow Institute of Physics and Technology | Potapenko A.P.,Moscow Institute of Physics and Technology | Borshchevskiy V.I.,Moscow Institute of Physics and Technology | And 9 more authors.
Doklady Biochemistry and Biophysics | Year: 2016

In humans, two endothelin receptors, ETa and ETb, are activated by three endogenous 21-mer cyclic peptides, ET-1, ET-2, and ET-3, which control various physiological processes, including vasoconstriction, vasodilation, and stimulation of cell proliferation. The first stage of this study it to produce a stable solubilized and purified receptor in a monodisperse state. This article is focused on the engineering, expression, purification, and characterization of the endothelin receptor B for subsequent structural and functional studies. © 2016, Pleiades Publishing, Ltd.

Nogly P.,New University of Lisbon | Gushchin I.,University Grenoble alpes | Gushchin I.,French National Center for Scientific Research | Gushchin I.,CEA Grenoble | And 26 more authors.
Nature Communications | Year: 2014

Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65⠉Šresolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane α-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed. © 2014 Macmillan Publishers Limited.

Kuklin A.I.,Joint Institute for Nuclear Research | Murugova T.N.,Joint Institute for Nuclear Research | Ivankov O.I.,Taras Shevchenko National University | Rogachev A.V.,Joint Institute for Nuclear Research | And 8 more authors.
Journal of Physics: Conference Series | Year: 2012

The results of small angle scattering investigation of protein apoferritin are presented. The sizes and shapes, including those determined by indirect Fourier transform method, are calculated. The pair-distance distribution function for both small angle neutron (SANS) and X-ray small angle scattering (SAXS) are obtained. It is shown that SANS and SAXS methods give similar shape (spherical shell with holes) of apoferritin. At the same time, fits of experimental data for SAXS and SANS curves give a little different sizes and volumes of the molecule. The reasons for these differences are discussed. © Published under licence by IOP Publishing Ltd.

Polovinkin V.,University Grenoble alpes | Polovinkin V.,French National Center for Scientific Research | Polovinkin V.,CEA Grenoble | Polovinkin V.,Moscow Institute of Physics and Technology | And 24 more authors.
Journal of Membrane Biology | Year: 2014

Amphipols (APols) have become important tools for the stabilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs solubilized in APols would be of high importance for structural biology. However, despite considerable efforts, it is still not clear whether MP/APol complexes can form well-ordered crystals suitable for X-ray crystallography. In the present work, we show that an APol-trapped MP can be crystallized in meso. Bacteriorhodopsin (BR) trapped by APol A8-35 was mixed with a lipidic mesophase, and crystallization was induced by adding a precipitant. The crystals diffract beyond 2 Å. The structure of BR was solved to 2 Å and found to be indistinguishable from previous structures obtained after transfer from detergent solutions. We suggest the proposed protocol of in meso crystallization to be generally applicable to APol-trapped MPs. © 2014, Springer Science+Business Media New York.

Polovinkin V.,University Grenoble alpes | Polovinkin V.,French National Center for Scientific Research | Polovinkin V.,CEA Grenoble | Polovinkin V.,Moscow Institute of Physics and Technology | And 22 more authors.
Journal of Membrane Biology | Year: 2014

Surface-enhanced Raman spectroscopy (SERS) has developed dramatically since its discovery in the 1970s, because of its power as an analytical tool for selective sensing of molecules adsorbed onto noble metal nanoparticles (NPs) and nanostructures, including at the single-molecule (SM) level. Despite the high importance of membrane proteins (MPs), SERS application to MPs has not really been studied, due to the great handling difficulties resulting from the amphiphilic nature of MPs. The ability of amphipols (APols) to trap MPs and keep them soluble, stable, and functional opens up onto highly interesting applications for SERS studies, possibly at the SM level. This seems to be feasible since single APol-trapped MPs can fit into gaps between noble metal NPs, or in other gap-containing SERS substrates, whereby the enhancement of Raman scattering signal may be sufficient for SM sensitivity. The goal of the present study is to give a proof of concept of SERS with APol-stabilized MPs, using bacteriorhodopsin (BR) as a model. BR trapped by APol A8-35 remains functional even after partial drying at a low humidity. A dried mixture of silver Lee–Meisel colloid NPs and BR/A8-35 complexes give rise to SERS with an average enhancement factor in excess of 102. SERS spectra resemble non-SERS spectra of a dried sample of BR/APol complexes. © 2014, Springer Science+Business Media New York.

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