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Gorshkova Y.E.,Joint Institute for Nuclear Research | Kuklin A.I.,Joint Institute for Nuclear Research | Gordeliy V.I.,RAS Institute for Nuclear Research | Gordeliy V.I.,Institute of Structural Biology
Journal of Surface Investigation | Year: 2017

Results obtained via small-angle neutron scattering studies of the influence of calcium ions on the structure and phase transitions of phospholipid membranes are presented. The main phase transition temperature of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (1 wt %) multilamellar vesicles is demonstrated to increase by more than 1°C even when the calcium-ion content of the solution is low (0.1 mM). Detailed analysis of the multilamellar vesicles transition between “bound” and “unbound” state indicates the continuous character of the investigated process in both liquid and gel phases. The critical Ca2+ ion concentrations which initiate the destruction of the multilamellar structures and the formation of unilamellar vesicles are found to be ~0.3 mM in the gel and ~0.4–0.5 mM in the liquid-crystal phases during heating and ~0.5 mM in the phases under study upon cooling. © 2017, Pleiades Publishing, Ltd.

Zalewski J.K.,University of Pittsburgh | Heber S.,University of Pittsburgh | Heber S.,Institute of Structural Biology | Mo J.H.,University of Pittsburgh | And 3 more authors.
Journal of Visualized Experiments | Year: 2017

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization. © 2017 Journal of Visualized Experiments.

Scarsdale J.N.,Institute of Structural Biology | Walavalkar N.M.,Virginia Commonwealth University | Buchwald W.A.,Virginia Commonwealth University | Ginder G.D.,University of North Carolina at Chapel Hill | Williams Jr. D.C.,University of North Carolina at Chapel Hill
Journal of Biological Chemistry | Year: 2014

Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Strulovich R.,Institute of Structural Biology | Attali B.,Tel Aviv University | Hirsch J.A.,Institute of Structural Biology | Hirsch J.A.,Tel Aviv University
Biochemistry | Year: 2016

The Kv7 (KCNQ) channel family, comprising voltage-gated potassium channels, plays major roles in fine-tuning cellular excitability by reducing firing frequency and controlling repolarization. Kv7 channels have a unique intracellular C-terminal (CT) domain bound constitutively by calmodulin (CaM). This domain plays key functions in channel tetramerization, trafficking, and gating. CaM binds to the proximal CT, comprising helices A and B. Kv7.2 and Kv7.3 are expressed in neural tissues. Together, they form the heterotetrameric M channel. We characterized Kv7.2, Kv7.3, and chimeric Kv7.3 helix A-Kv7.2 helix B (Q3A-Q2B) proximal CT/CaM complexes by solution methods at various Ca2+concentrations and determined them all to have a 1:1 stoichiometry. We then determined the crystal structure of the Q3A-Q2B/CaM complex at high Ca2+ concentration to 2.0 Å resolution. CaM hugs the antiparallel coiled coil of helices A and B, braced together by an additional helix. The structure displays a hybrid apo-Ca2+ CaM conformation even though four Ca2+ ions are bound. Our results pinpoint unique interactions enabling the possible intersubunit pairing of Kv7.3 helix A and Kv7.2 helix B while underlining the potential importance of Kv7.3 helix A's role in stabilizing channel oligomerization. Also, the structure can be used to rationalize various channelopathic mutants. Functional testing of the chimeric channel found it to have a voltage-dependence similar to the M channel, thereby demonstrating helix A's importance in imparting gating properties. © 2016 American Chemical Society.

Gargallo R.,University of Barcelona | Eritja R.,Institute of Structural Biology | Kudrev A.G.,Saint Petersburg State University
Russian Journal of General Chemistry | Year: 2010

UV absorption spectra and circular dichroism spectra of aqueous solutions of cytosin- and thyminecontaining single-stranded Oligodeoxyribonucleotide 5′-CCTTTCCTTTTCCTTTCC-3′(ckit4) were measured at various pH in the range 3.3-8.9. The chemometric analysis of the multiinstrumental data matrix was carrie out. The diagrams of relative contents of complex forms of the DNA molecule absorbing in the studied wavelength range (220-320 nm) were constructed by the ALS-MCR soft simulation procedure without initial postulation of their chemical compositions. The model of equilibrium complex formation describing observed changes in the spectra depending on the solution acidity was developed on the basis of the matrix method. Intrinsic protonation constants of the oligonucleotide ckit4 were calculated. The formation of intramolecular complexes between cytosine C.C+ bases in the studied DNA molecule is of a cooperative nature, and thei subsequent protonation is an anticooperative process. © 2010 Pleiades Publishing, Ltd.

Sachyani D.,Institute of Structural Biology | Dvir M.,Tel Aviv University | Strulovich R.,Institute of Structural Biology | Tria G.,German Electron Synchrotron | And 7 more authors.
Structure | Year: 2014

Kv7 channels tune neuronal and cardiomyocyte excitability. In addition to the channel membrane domain, they also have a unique intracellular C-terminal (CT) domain, bound constitutively to calmodulin (CaM). This CT domain regulates gating and tetramerization. We investigated the structure of the membrane proximal CT module in complex with CaM by X-ray crystallography. The results show how the CaM intimately hugs a two-helical bundle, explaining many channelopathic mutations. Structure-based mutagenesis of this module in the context of concatemeric tetramer channels and functional analysis along with in vitro data lead us to propose that one CaM binds to one individual protomer, without crosslinking subunits and that this configuration is required for proper channel expression and function. Molecular modeling of the CT/CaM complex in conjunction with small-angle X-ray scattering suggests that the membrane proximal region, having a rigid lever arm, is a critical gating regulator. © 2014 Elsevier Ltd.

Dames S.A.,TU Munich | Dames S.A.,Helmholtz Center Munich | Dames S.A.,Institute of Structural Biology
Journal of Peptide Science | Year: 2015

The nematocyst walls of Hydra are formed by proteins containing small cysteine-rich domains (CRDs) of ~25 amino acids. The first CRD of nematocyst outer all antigen (NW1) and the C-terminal CRD of minicollagen-1 (Mcol1C) contain six cysteines at identical sequence positions, however adopt different disulfide bonded structures. NW1 shows the disulfide connectivities C2-C14/C6-C19/C10-C18 and Mcol1C C2-C18/C6-C14/C10-C19. To analyze if both show structural preferences in the open, non-disulfide bonded form, which explain the formation of either disulfide connectivity pattern, molecular dynamics (MD) simulations at different temperatures were performed. NW1 maintained in the 100-ns MD simulations at 283 K a rather compact fold that is stabilized by specific hydrogen bonds. The Mcol1C structure fluctuated overall more, however stayed most of the time also rather compact. The analysis of the backbone Φ/ψ angles indicated different turn propensities for NW1 and Mcol1C, which mostly can be explained based on published data about the influence of different amino acid side chains on the local backbone conformation. Whereas a folded precursor mechanism may be considered for NW1, Mcol1C may fold according to the quasi-stochastic folding model involving disulfide bond reshuffling and conformational changes, locking the native disulfide conformations. The study further demonstrates the power of MD simulations to detect local structural preferences in rather dynamic systems such as the open, non-disulfide bonded forms of NW1 and Mcol1C, which complement published information from NMR backbone residual dipolar couplings. Because the backbone structural preferences encoded by the amino acid sequence embedding the cysteines influence which disulfide connectivities are formed, the data are generally interesting for a better understanding of oxidative folding and the design of disulfide stabilized therapeutics. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Kuklin A.I.,Joint Institute for Nuclear Research | Rogov A.D.,Joint Institute for Nuclear Research | Gorshkova Y.E.,Joint Institute for Nuclear Research | Utrobin P.K.,Moscow Institute of Physics and Technology | And 7 more authors.
Physics of Particles and Nuclei Letters | Year: 2011

The results of experimental and computer-modeling investigations of neutron spectra and fluxes obtained with cold and thermal moderators at the IBR-2 reactor (Joint Institute for Nuclear Research (JINR), Dubna) are presented. These studies are for the YuMO small-angle neutron scattering (SANS) spectrometer (IBR-2 beamline 4). The neutron spectra have been measured for two methane cold moderators for the standard configuration of the SANS instrument. The data from both moderators under different conditions of their operation are compared. The ratio of experimentally determined neutron fluxes of cold and thermal moderators is shown at different wavelengths. Monte Carlo simulations have been carried out to determine the spectra for cold-methane and thermal moderators. The results of calculations of the ratio of neutron fluxes of cold and thermal moderators at different wavelengths are demonstrated. In addition, the absorption of neutrons in the air gaps on the way from the moderator to the investigated sample is presented. SANS with the protein apoferritin was done with both cold methane and a thermal moderator and the data were compared. The prospects for the use of a cold moderator for a SANS spectrometer at IBR-2 are discussed. The advantages of using the YuMO spectrometer with a thermal moderator with respect to the tested cold moderator are shown. © 2011 Pleiades Publishing, Ltd.

Holton S.J.,German Electron Synchrotron | Anandhakrishnan M.,German Electron Synchrotron | Geerlof A.,German Electron Synchrotron | Geerlof A.,Institute of Structural Biology | Wilmanns M.,German Electron Synchrotron
Journal of Structural Biology | Year: 2013

Hydroxyacid dehydrogenases, responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids in lactic acid producing bacteria, have a range of biotechnology applications including antibiotic synthesis, flavor development in dairy products and the production of valuable synthons. The genome of Lactobacillus delbrueckii ssp. bulgaricus, a member of the heterogeneous group of lactic acid bacteria, encodes multiple hydroxyacid dehydrogenases whose structural and functional properties remain poorly characterized. Here, we report the apo and coenzyme NAD+ complexed crystal structures of the L. bulgaricus D-isomer specific 2-hydroxyacid dehydrogenase, D2-HDH. Comparison with closely related members of the NAD-dependent dehydrogenase family reveals that whilst the D2-HDH core fold is structurally conserved, the substrate-binding site has a number of non-canonical features that may influence substrate selection and thus dictate the physiological function of the enzyme. © 2012 Elsevier Inc.

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