26 research outputs found

    NMR solution structure of TbCof.

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    <p>A. The lowest-energy conformation was used for the cartoon representation of TbCof, showing a central β sheet surrounded by α helices. The key secondary structure elements, both termini, and the F-loop are labeled; B. Superposition of backbone traces of the 20 lowest-energy NMR structures of TbCof. C. Electrostatic surface diagram of the lowest-energy conformation of TbCof is shown from two different orientations 180 degrees apart (red, negative; blue, positive; white, neutral).</p

    Interactions between TbCof and G-actin revealed by ITC.

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    <p>The negative peaks indicate an exothermic reaction. The area under each peak represents the heat released after an injection of TbCof into G-actin solution (upper panel). Binding isotherms were obtained by plotting the peak areas against the molar ratio of TbCof to G-actin (lower panel). The lines represent the best-fit curves obtained from least-squares regression analyses assuming a one-site binding model. A. ITC of TbCof-G-actin in the presence of ADP. B. ITC of TbCof-G-actin in the presence of ATP.</p

    Structural comparison of TbCof with other ADF/cofilin family members.

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    <p>A. ADF/cofilin from <i>Trypanosoma brucei</i>; B. cofilin from <i>Saccharomyces cerevisiae</i> (PDB ID: 1COF); C. ADF1 from <i>Plasmodium falciparum</i> (PfADF1) (PDB ID: 2XF1); D. ADF2 from <i>Plasmodium berghei</i> (PbADF2) (PDB ID: 2XFA). These ADF/cofilin family members all share the classical fold except for a short helical turn in the loop between β6 and the C-terminal helix from residue D119 to L123 in TbCof and the shorter C-terminal region in PfADF1. The key secondary structure elements are labeled.</p

    Model of TbCof (cyan) in complex with G-actin (magenta).

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    <p>The G-actin binding site contains 3 regions: a, the N-terminal extension that interacts with actin subdomain 1; b, the long kinked helix α3 that binds to the cleft between actin subdomains 1 and 3; c, the region before the C-terminal α-helix that interacts with actin subdomain 3. F-actin binding is mediated by additional regions consisting of the F-loop between β4-β5 (with label d) and the C-terminal α-helix (with label e). The F-actin binding site is circled by a dashed line.</p

    Effect of TbCof on F-actin examined by electron microscopy.

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    <p>F-actin (5 µM) was incubated without (A) or with 0.05 µM TbCof (B), negatively stained with uranyl acetate, and observed by electron microscopy. Actin alone maintains long filaments. While only short filaments are observed in the presence of TbCof (B). The scale bars represent 100 nm.</p

    Structural and Functional Insight into ADF/Cofilin from <em>Trypanosoma brucei</em>

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    <div><p>The ADF/cofilin family has been characterized as a group of actin-binding proteins critical for controlling the assembly of actin within the cells. In this study, the solution structure of the ADF/cofilin from <em>Trypanosoma brucei</em> (TbCof) was determined by NMR spectroscopy. TbCof adopts the conserved ADF/cofilin fold with a central β-sheet composed of six β-strands surrounded by five α-helices. Isothermal titration calorimetry experiments denoted a submicromolar affinity between TbCof and G-actin, and the affinity between TbCof and ADP-G-actin was five times higher than that between TbCof and ATP-G-actin at low ionic strength. The results obtained from electron microscopy and actin filament sedimentation assays showed that TbCof depolymerized but did not co-sediment with actin filaments and its ability of F-actin depolymerization was pH independent. Similar to actin, TbCof was distributed throughout the cytoplasm. All our data indicate a structurally and functionally conserved ADF/cofilin from <em>Trypanosoma brucei</em>.</p> </div

    Localization of TbCof in the procylic form <i>T. brucei</i>.

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    <p>Cells overexpressing TbCof with an HA<sub>3</sub>-tag at the C-terminus were treated with tetracycline (1 µg/ml) for 2 days. (A) The overexpressed TbCof-HA<sub>3</sub> examined by western blot using an HA probe. (B) Cells overexpressing TbCof-HA<sub>3</sub> stained with an HA probe and DAPI, and examined with a fluorescence microscope. TbCof is localized to the cytoplasm throughout the cell cycle.</p

    Sequence alignment of TbCof with other ADF/cofilin members.

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    <p>The alignment was prepared using ClustalW2 and ESPript 2.2. Identical residues are boxed in red. The accession numbers of the proteins used for the alignment are listed as follows: Swiss-Prot, Q2QKR1, ADF/cofilin from <i>Leishmania donovani</i> (LdCof); Swiss-Prot, B9Q2C8, ADF from <i>Toxoplasma gondii</i> (TgADF); Swiss-Prot, P86292, ADF1 from <i>Plasmodium falciparum</i> (PfADF1); Swiss-Prot, Q4YT54, ADF2 from <i>Plasmodium berghei</i> (PbADF2); Swiss-Prot, P23528, cofilin from <i>Homo sapiens</i> (HuCof)<i>;</i> Swiss-Prot, Q39250, ADF1 from <i>Arabidopsis Thaliana</i> (AtADF1); Swiss-Prot, Q03048, cofilin from <i>Saccharomyces cerevisiae</i> (ScCof). The secondary structure elements of TbCof are labeled on the top of the alignment.</p

    Host–Guest Chemistry of Dendrimer–Drug Complexes: 7. Formation of Stable Inclusions between Acetylated Dendrimers and Drugs Bearing Multiple Charges

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    Drug molecules bearing multiple charges usually form precipitates with cationic dendrimers, which presents a challenge during the preparation of dendrimer inclusions for these drugs. In the present study, fully acetylated polyamidoamine (PAMAM) dendrimers were proposed as stable vehicles for drug molecules bearing two negative charges such as Congo red and indocyanine green. NMR techniques including <sup>1</sup>H NMR and <sup>1</sup>H-<sup>1</sup>H NOESY were used to characterize the host–guest chemistry of acetylated dendrimer and these guest molecules. The cationic PAMAM dendrimer was found to form a precipitate with Congo red and indocyanine green, but the acetylated one avoided the formation of cross-linking structures in aqueous solutions. NOESY studies revealed the encapsulation of Congo red and indocyanine green within the interior cavities of PAMAM dendrimers at mild acidic conditions and acetylated dendrimers show much stronger ability to encapsulate the guest molecules than cationic ones. Also, UV–vis–NIR studies suggest that acetylated dendrimers significantly improve the photostability of indocyanine green and prevent the formation of indocyanine green J-aggregates in aqueous solutions. The present study provides a new insight into dendrimer-based host–guest systems, especially for those guest molecules bearing multiple charges
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