7 research outputs found

    Unfolding of Fha30 using simulated molecular dynamics.

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    <p>(A) Comparison of a typical experimental force curve with a simulated F-D unfolding curve. Steered molecular dynamics simulation was performed using a coarse-grained model of the protein structure without N- and C-terminal tags (304 residues). The colored arrows indicate the positions along the simulation where contact maps were calculated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073572#pone.0073572.s002" target="_blank">Fig. S2</a> in Supporting information). (B) Representation of the Fha30 structure showing the structural elements (colored according to the arrows in panel A) that unfolded in the successive force peaks. (C) Series of snapshots along the SMD unfolding trajectory. The structural elements are colored according to the events observed in the F-D curve and numbered in panel in A. The colored arrows indicate the regions of the protein that unfolded in each of the successive force peaks.</p

    Unfolding of Fha60 by single-molecule AFM.

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    <p>(A) Experimental set-up. (B) Representative force-distance curves obtained by stretching single polypeptides, showing periodic features reflecting sequential unfolding peaks. Force peaks were well described by the WLC model (red line with Lc (nm)), using a persistence length of 0.4 nm. The curves shown are representative of a total of more than 200 adhesives curves obtained using 5 independent tips and 5 sample preparations. (C) Superposition of 30 typical force curves showing that the last six force peaks in particular are reproducibly observed. (D) Histograms of contour length Lc (n = 171) of the different peaks with Gaussian fit and statistics (mean ± SD). (E,F) Bivariate color-coded contour plots of Lc-ΔLc pairs for every individual unfolding event. Blue and red represent low and high frequencies of events, respectively.</p

    Schematic representation of FHA and model proteins used in this work.

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    <p>(A) Model of FHA. The dotted vertical lines at the right side of the protein model indicate the approximate lengths of Fha60 and Fha30, which both have the same N-terminus as full-length FHA. The TPS domain is shown in blue, the R1 region is shown in red, and the B1, R2 and B2 domains are shown in green, magenta and cyan, respectively. The X-ray structure of Fha30 is known, while the R1 and R2 regions were built by using the models reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073572#pone.0073572-Kajava3" target="_blank">[18]</a>, and the B1 and B2 regions were built by molecular modeling using the I-TASSER web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073572#pone.0073572-Roy1" target="_blank">[19]</a>. (B) Conserved regions of Fha30. On the basis of multiple sequence alignments, four regions of different conservation rates were identified in the TPS domain. The most conserved subdomains C1 and C2 are shown in dark blue, and the less conserved subdomains LC1 and LC2 are shown in light blue. The first R1 coils are in red. (C) Structural organization of Fha30. The N-terminal cap and the six successive coils (Coil_A to Coil_F) of the TPS domain are shown in magenta, blue, cyan, green, yellow, orange and brown, respectively. The three R1 coils present in the crystal structure of Fha30 are shown in dark red, red and pink, and the three extra-helical elements are shown in grey. Elements II and III assemble together to form a four-stranded βsheet that packs against the β-helical core formed by coils_A to _F.</p

    Localizing Chemical Groups while Imaging Single Native Proteins by High-Resolution Atomic Force Microscopy

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    Simultaneous high-resolution imaging and localization of chemical interaction sites on single native proteins is a pertinent biophysical, biochemical, and nanotechnological challenge. Such structural mapping and characterization of binding sites is of importance in understanding how proteins interact with their environment and in manipulating such interactions in a plethora of biotechnological applications. Thus far, this challenge remains to be tackled. Here, we introduce force–distance curve-based atomic force microscopy (FD-based AFM) for the high-resolution imaging of SAS-6, a protein that self-assembles into cartwheel-like structures. Using functionalized AFM tips bearing Ni<sup>2+</sup>-<i>N</i>-nitrilotriacetate groups, we locate specific interaction sites on SAS-6 at nanometer resolution and quantify the binding strength of the Ni<sup>2+</sup>-NTA groups to histidine residues. The FD-based AFM approach can readily be applied to image any other native protein and to locate and structurally map histidine residues. Moreover, the surface chemistry used to functionalize the AFM tip can be modified to map other chemical interaction sites

    Mechanical Forces Guiding Staphylococcus aureus Cellular Invasion

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    Staphylococcus aureus can invade various types of mammalian cells, thereby enabling it to evade host immune defenses and antibiotics. The current model for cellular invasion involves the interaction between the bacterial cell surface located fibronectin (Fn)-binding proteins (FnBPA and FnBPB) and the α5β1 integrin in the host cell membrane. While it is believed that the extracellular matrix protein Fn serves as a bridging molecule between FnBPs and integrins, the fundamental forces involved are not known. Using single-cell and single-molecule experiments, we unravel the molecular forces guiding S. aureus cellular invasion, focusing on the prototypical three-component FnBPA–Fn–integrin interaction. We show that FnBPA mediates bacterial adhesion to soluble Fn <i>via</i> strong forces (∼1500 pN), consistent with a high-affinity tandem β-zipper, and that the FnBPA–Fn complex further binds to immobilized α5β1 integrins with a strength much higher than that of the classical Fn–integrin bond (∼100 pN). The high mechanical stability of the Fn bridge favors an invasion model in which Fn binding by FnBPA leads to the exposure of cryptic integrin-binding sites <i>via</i> allosteric activation, which in turn engage in a strong interaction with integrins. This activation mechanism emphasizes the importance of protein mechanobiology in regulating bacterial–host adhesion. We also find that Fn-dependent adhesion between S. aureus and endothelial cells strengthens with time, suggesting that internalization occurs within a few minutes. Collectively, our results provide a molecular foundation for the ability of FnBPA to trigger host cell invasion by S. aureus and offer promising prospects for the development of therapeutic approaches against intracellular pathogens
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