Institute of Complex Systems ICS

Germany

Institute of Complex Systems ICS

Germany
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Zander U.,European Synchrotron Radiation Facility | Bourenkov G.,European Molecular Biology Laboratory | Popov A.N.,European Synchrotron Radiation Facility | De Sanctis D.,European Synchrotron Radiation Facility | And 15 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2015

Here, an automated procedure is described to identify the positions of many cryocooled crystals mounted on the same sample holder, to rapidly predict and rank their relative diffraction strengths and to collect partial X-ray diffraction data sets from as many of the crystals as desired. Subsequent hierarchical cluster analysis then allows the best combination of partial data sets, optimizing the quality of the final data set obtained. The results of applying the method developed to various systems and scenarios including the compilation of a complete data set from tiny crystals of the membrane protein bacterio rhodopsin and the collection of data sets for successful structure determination using the single-wavelength anomalous dispersion technique are also presented. © 2015 International Union of Crystallography.


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.


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.


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.


Lange W.,Institute of Complex Systems ICS | Jin L.,Institute of Complex Systems ICS | Maybeck V.,Institute of Complex Systems ICS | Meisenberg A.,Jülich Research Center | And 2 more authors.
Progress in Biomedical Optics and Imaging - Proceedings of SPIE | Year: 2014

Genetically encoded light-sensitive proteins can be used to manipulate and observe cellular functions. According to different modes of action, these proteins are divided into actuators like the blue-light gated cation channel Channelrhodopsin-2 (ChR2) and detectors like the calcium sensor GCaMP. In order to optogenetically control and study the activity of rat primary cortical neurons, we established a transduction procedure using recombinant Adeno-associated viruses (rAAVs) as gene-ferries. Thereby, we achieved high transduction rates of these neurons with ChR2. In ChR2 expressing neurons, action potentials could be repeatedly and precisely elicited with laser pulses and measured via patch clamp recording. © 2014 SPIE.


Ishchenko A.,Institute of Complex Systems ICS | Ishchenko A.,RWTH Aachen | Round E.,CNRS Institute of Pharmacology and Structural Biology | Round E.,French National Center for Scientific Research | And 24 more authors.
Journal of Photochemistry and Photobiology B: Biology | Year: 2013

The complex of sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII) mediates negative phototaxis in halobacteria Natronomonas pharaonis. Upon light activation NpSRII triggers, by means of NpHtrII, a signal transduction chain homologous to the two component system in eubacterial chemotaxis. Here we report on the crystal structure of the ground state of the mutant NpSRII-D75N/NpHtrII complex in the space group I212 121. Mutations of this aspartic acid in light-driven proton pumps dramatically modify or/and inhibit protein functions. However, in vivo studies show that the similar D75N mutation retains functionality of the NpSRII/NpHtrII complex. The structure provides the molecular basis for the explanation of the unexpected observation that the wild and the mutant complexes display identical physiological response on light excitation. © 2013 Elsevier B.V. All rights reserved.


Borshchevskiy V.I.,CNRS Institute of Pharmacology and Structural Biology | Borshchevskiy V.I.,Institute of Complex Systems ICS | Borshchevskiy V.I.,Moscow Institute of Physics and Technology | Round E.S.,CNRS Institute of Pharmacology and Structural Biology | And 7 more authors.
Journal of Molecular Biology | Year: 2011

Bacteriorhodopsin (bR) provides light-driven vectorial proton transport across a cell membrane. Creation of electrochemical potential at the membrane is a universal step in energy transformation in a cell. Published atomic crystallographic models of early intermediate states of bR show a significant difference between them, and conclusions about pumping mechanisms have been contradictory. Here, we present a quantitative high-resolution crystallographic study of conformational changes in bR induced by X-ray absorption. It is shown that X-ray doses that are usually accumulated during data collection for intermediate-state studies are sufficient to significantly alter the structure of the protein. X-ray-induced changes occur primarily in the active site of bR. Structural modeling showed that X-ray absorption triggers retinal isomerization accompanied by the disappearance of electron densities corresponding to the water molecule W402 bound to the Schiff base. It is demonstrated that these and other X-ray-induced changes may mimic functional conformational changes of bR leading to misinterpretation of the earlier obtained X-ray crystallographic structures of photointermediates. © 2011 Elsevier Ltd. All rights reserved.


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.

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