7 research outputs found

    Hybrid Approach for Highly Coarse-Grained Lipid Bilayer Models

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    We present a systematic methodology to develop highly coarse-grained (CG) lipid models for large scale biomembrane simulations, in which we derive CG interactions using a powerful combination of the multiscale coarse-graining (MS-CG) method, and an analytical form of the CG potential to model interactions at short-range. The resulting hybrid coarse-graining (HCG) methodology is used to develop a three-site solvent-free model for 1,2-dilauroyl-<i>sn</i>-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine (DOPC), and a 1:1 mixture of 1,2-dioleoyl-<i>sn</i>-glycero-3-phospho-l-serine (DOPS) and DOPC. In addition, we developed a four-site model of DOPC, demonstrating the capability of the HCG methodology in designing model lipid systems of a desired resolution. We carried out microsecond-scale molecular dynamics (MD) simulations of large vesicles, highlighting the ability of the model to study systems at mesoscopic length and time scales. The models of DLPC, DOPC, and DOPC/DOPS have elastic properties consistent with experiment and structural properties such as the radial distribution functions (RDF), bond and angle distributions, and the <i>z</i>-density distributions that compare well with reference all-atom systems

    Mechanistic Insights into the Differential Catalysis by RheB and Its Mutants: Y35A and Y35A-D65A

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    RheB GTPase is a Ras-related molecular switch, which regulates the mTOR signaling pathway by cycling between the active [guanosine triphosphate (GTP)] state and inactive [guanine diphosphate (GDP)] state. Impairment of GTPase activity because of mutations in several small GTPases is known to be associated with several cancers. The conventional GTPase mechanism such as in H-Ras requires a conserved glutamine (Q64) in the switch-II region of RheB to align the catalytic water molecule for efficient GTP hydrolysis. The conformation of this conserved glutamine is different in RheB, resulting in an altered conformation of the entire switch-II region. Studies on the atypical switch-II conformation in RheB revealed a distinct, noncanonical mode of GTP hydrolysis. An RheB mutant Y35A was previously shown to exclusively enhance the intrinsic GTPase activity of RheB, whereas the Y35A-D65A double mutant was shown to reduce the elevated GTPase activity. Here, we have used all-atom molecular dynamics (MD) simulations for comprehensive understanding of the conformational dynamics associated with the fast (Y35A) and slow (Y35A-D65A) hydrolyzing mutants of RheB. Using a combination of starting models from PDB structures and in-silico generated mutant structures, we discuss the observed conformational deviations in wild type (WT) versus mutants. Our results show that a number of interactions of RheB with phosphates of GTP as well as Mg<sup>2+</sup> are destabilized in Y35A mutant in the switch-I region. We report distinct water dynamics at the active site of WT and mutants. Furthermore, principal component analysis showed significant differences in the conformational space sampled by the WT and mutants. Our observations provide improved understanding of the noncanonical GTP hydrolysis mechanism adopted by RheB and its modulation by Y35A and Y35A-D65A mutants

    K<sub>D</sub> for var2CSA recombinant proteins binding to placental CSPG.

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    <p>K<sub>D</sub> values were determined from the concentration dependence of steady-state SPR response using the Biacore BIAEVALUATION 3.1 software.</p><p>*: from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone.0020270-Srivastava1" target="_blank">[34]</a>.</p>#<p>: from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone.0020270-Gangnard1" target="_blank">[41]</a>.</p

    Depletion of purified adhesion-inhibitory rabbit IgGs (anti 3D7-DBL1X-6ε) by various recombinant proteins.

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    <p>Immunization of rabbit with 3D7-DBL1X-6ε induces adhesion-blocking antibodies. Purified IgG against 3D7-DBL1X-6ε (Anti-DBL1X-6ε was depleted on 3D7-DBL1X, 3D7-DBL2X, FCR3-DBL3X, 3D7-DBL5ε, 3D7-DBL6ε, 3D7-DBL1X-2X, 3D7-DBL1X-CIDR, FCR3-DBL3X-4ε, 3D7-DBL1X-3X, 3D7-DBL1X-4ε, 3D7-DBL1X-5ε and 3D7-DBL1X-6ε immobilized on tosylactivated beads. Unbound (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone-0020270-g005" target="_blank">Fig. 5A</a>) and bound (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020270#pone-0020270-g005" target="_blank">Fig. 5B</a>) fractions were tested for inhibition of binding of 3D7-DBL1X-6ε to decorin. Data are express as % of inhibition compare to control.</p

    Various var2CSA recombinant proteins expressed in HEK293 cells and <i>E. coli</i>.

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    <p>(A). Schematic view of the var2CSA domain organization and sequence limits of the recombinant domains studied (3D7-DBL1X, 3D7-DBL2X, FCR3-DBL3X, 3D7-DBL5ε, 3D7-DBL1X-2X, FCR3-DBL3X-4ε, 3D7-DBL1X-CIDR, 3D7-DBL1X-3X, 3D7-DBL1X-4ε, 3D7-DBL1X-5ε and 3D7-DBL1X-6ε). Var2CSA comprises six DBL domains (DBL1X to DBL6ε), a CIDRpam domain and three inter-domain regions (INT1-3) in the extracellular region, together with a trans-membrane segment and acidic C terminus sequence (ATS). DBL1X, DBL2X, DBL3X, DBL4ε, DBL5ε and DBL6ε are shown in green; CIDR in orange; N-terminal sequence (NTS) and inter-domain regions (INT) in grey; the trans-membrane and ATS regions in blue. The length of each bar corresponds to the domain size. (B). Purification of var2CSA derived proteins expressed in HEK293 and <i>E. coli</i>. Nu-SDS-PAGE Precast 4–12% Bis-Tris gel under nonreducing (B) and reducing (C) conditions was loaded with purified recombinant proteins. Lane 1: Marker, lane 2: 3D7-DBL1X, lane 3: 3D7-DBL2X, lane 4: FCR3-DBL3X, lane 5: 3D7-DBL5ε, lane 6: 3D7-DBL1X-2X, lane 7: FCR3-DBL3X-4ε, lane 8: 3D7-DBL1X-CIDR, lane 9: 3D7-DBL1X-3X, lane 10: 3D7-DBL1X-4ε, lane 11: 3D7-DBL1X-5ε and lane 12: 3D7-DBL1X-6ε. Proteins were visualized with Coomassie blue.</p

    Competitive inhibition of recombinant DBL domains binding to decorin using various glycosaminoglycans.

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    <p>Recombinant proteins (A) 3D7-DBL1X-2X, (B) 3D7-DBL1X-CIDR, (C) 3D7-DBL1X-3X, (D) 3D7-DBL1X-4ε, (E) 3D7-DBL1X-5ε and (E) 3D7-DBL1X-6ε at 1 µg/mL were premixed with increasing amounts of BSA (□) or glycosaminoglycans, 0.156–100 µg/mL of CSA (×), CSC (⋄) or HS (▵) and incubated in plates previously coated with decorin.</p

    Binding of recombinant DBL domains from 3D7 and FCR3-DBL1X-6ε to different glycosaminoglycans.

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    <p>ELISA-based direct binding assay was performed to identify the specificity of (A) 3D7-DBL2X, (B) FCR3-DBL3X, (C) 3D7-DBL5ε, (D) FCR3-DBL3X-4ε, (E) 3D7-DBL1X-2X, (F) 3D7-DBL1X-CIDR, (G) 3D7-DBL1X-3X, (H) 3D7-DBL1X-4ε, (I) 3D7-DBL1X-5ε and (J) 3D7-DBL1X-6ε to different sulfated glycosaminoglycans. Increasing concentrations of recombinant proteins at serial dilutions of 0.31–20 µg/mL were added to wells previously coated with BSA (▵) or different glycosaminoglycans: decorin (○), CSA (□), CSC (⋄), HS (×).</p
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