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Huang G.Y.,Baylor College of Medicine | Kim J.J.,Baylor College of Medicine | Reger A.S.,Baylor College of Medicine | Lorenz R.,University of Kassel | And 11 more authors.
Structure | Year: 2014

Cyclic guanosine monophosphate (cGMP) and cyclic AMP (cAMP)-dependent protein kinases (PKG and PKA) are closely related homologs, and the cyclic nucleotide specificity of each kinase is crucial for keeping the two signaling pathways segregated, but the molecular mechanism of cyclic nucleotide selectivity is unknown. Here, we report that the PKG Iβ C-terminal cyclic nucleotide binding domain (CNB-B) is highly selective for cGMP binding, and we have solved crystal structures of CNB-B with and without bound cGMP. These structures, combined with a comprehensive mutagenic analysis, allowed us to identify Leu296 and Arg297 as key residues that mediate cGMP selectivity. In addition, by comparing the cGMP bound and unbound structures, we observed large conformational changes in the C-terminal helices in response to cGMP binding, which were stabilized by recruitment of Tyr351 as a "capping residue" for cGMP. The observed rearrangements of the C-terminal helices provide a mechanical insight into release of the catalytic domain and kinase activation. © 2014 Elsevier Ltd All rights reserved. Source

Patel T.R.,University of Manitoba | Morris G.A.,University of Nottingham | Zwolanek D.,University of Cologne | Keene D.R.,Shriners Hospital for Children | And 4 more authors.
Matrix Biology | Year: 2010

Laminins are multidomain glycoproteins that play important roles in development and maintenance of the extracellular matrix via their numerous interactions with other proteins. Several receptors for the laminin short arms revealed their importance in network formation and intercellular signaling. However, both the detailed structure of the laminin γ-1 short arm and its organization within the complexes is poorly understood due to the complexity of the molecule and the lack of a high-resolution structure. The presented data provide the first subatomic resolution structure for the laminin γ-1 short arm in solution. This was achieved using an integrated approach that combined a number of complementary biophysical techniques such as small angle X-ray scattering (SAXS), analytical ultracentrifugation, dynamic light scattering and electron microscopy. As a result of this study, we have obtained a significantly improved model for the laminin γ-1 short arm that represents a major step forward in molecular understanding of laminin-mediated complex formations. © 2010. Source

Barysheva M.M.,RAS Institute for Physics of Microstructures | Gribkov B.A.,RAS Institute for Physics of Microstructures | Vainer Yu.A.,RAS Institute for Physics of Microstructures | Zorina M.V.,RAS Institute for Physics of Microstructures | And 5 more authors.
Proceedings of SPIE - The International Society for Optical Engineering | Year: 2011

The requirements for multilayered x-ray elements for diffraction quality imaging optics (EUV - lithography, x-ray microscopy) achieves 0.2-0.3 nm roughness in spatial frequency range 10-3 - 103 mcm -1; it's also true for the substrates. Although, there are plenty of publications on studying a surface, when it comes to angstrom-quality substrates there is still a problem. In some cases we observe, standard methods like x-ray diffuse scattering (XRDS), atomic force microscopy (AFM) and optical interferometric microscopy (OIM) give notably different results in surface characterization. The goal of the attestation procedure is choosing the sample for sputtering a multilayer coating with better reflection properties, that's why it's important to understand the physical causes of the difference and get reliable information about the surface. In this work we discuss the limitation for aforesaid standard methods. OIM is seems to be inapplicable for supersmooth surface investigation because of applying references. It's also shown, that examination substrates with damaged layers in the volume (caused, for example, by ion-beam etching) by XRDS can lead to incorrect results. Imaging systems are composed by nonplanar optical elements with radiuses from 10 mm to 1 meter. That makes impossible using hard x-rays and also limited AFM applicability to high frequencies. Therefore, we propose the diffuse scattering of soft x-rays as an alternative approach. We also describe a new reflectometer, based on soft x-ray and visible light diffuse scattering, which can be used for surface investigation in middle and high spatial frequency ranges for both plane or curved substrates. © 2011 Copyright Society of Photo-Optical Instrumentation Engineers (SPIE). Source

Patel T.R.,University of Manitoba | Meier M.,University of Manitoba | Li J.,Rigaku Americas | Morris G.,University of Nottingham | And 3 more authors.
Protein Science | Year: 2011

Agrin is a large heparin sulphate proteoglycan with multiple domains, which is located in the extracellular matrix. The C-terminal G3 domain of agrin is functionally one of the most important domains. It harbors an α-dystroglycan binding site and carries out acetylcholine receptor clustering activities. In the present study, we have fused the G3 domain of agrin to an IgG Fc domain to produce a G3-Fc fusion protein that we intend to use as a tool to investigate new binding partners of agrin. As a first step of the study, we have characterized the recombinant fusion protein using a multidisciplinary approach using dynamic light scattering, analytical ultracentrifugation and small angle X-ray scattering (SAXS). Interestingly, our SAXS analysis using the high-resolution structures of G3 and Fc domain as models indicates that the G3-Fc protein forms a T-shaped molecule with the G3 domains extruding perpendicularly from the Fc scaffold. To validate our models, we have used the program HYDROPRO to calculate the hydrodynamic properties of the solution models. The calculated values are in excellent agreement with those determined experimentally. © 2011 The Protein Society. Source

Susner M.A.,Ohio State University | Susner M.A.,Oak Ridge National Laboratory | Sumption M.D.,Ohio State University | Takase A.,Rigaku Americas | Collings E.W.,Ohio State University
Superconductor Science and Technology | Year: 2014

In an investigation of possible atomic substitution for the Mg site in MgB2, superconducting thin films were deposited by pulsed laser deposition using MgB2 and ZrB2 targets. The resulting c-axis-oriented thin films contained various concentrations of Zr. The structural, chemical, and superconductive properties of these films were investigated. ZrB2 additions were found to increase the a lattice parameter; STEM-based chemical analysis showed Zr to be present within the grains. The superconducting critical temperature was suppressed for the heavily-doped samples. These observations are strong evidence for the substitution of Zr for Mg in the Mg sublattice of MgB2. © 2014 IOP Publishing Ltd. Source

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