6 research outputs found

    Label-Free, Multiplexed, Single-Molecule Analysis of Protein–DNA Complexes with Nanopores

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    Protein interactions with specific DNA sequences are crucial in the control of gene expression and the regulation of replication. Single-molecule methods offer excellent capabilities to unravel the mechanism and kinetics of these interactions. Here, we develop a nanopore approach where a target DNA sequence is contained in a hairpin followed by a ssDNA. This system allows DNA–protein complexes to be distinguished from bare DNA molecules as they are pulled through a single nanopore detector, providing both equilibrium and kinetic information. We show that this approach can be used to test the inhibitory effect of small molecules on complex formation and their mechanisms of action. In a proof of concept, we use DNAs with different sequence patterns to probe the ability of the nanopore to distinguish the effects of an inhibitor in a complex mixture of target DNAs and proteins. We anticipate that the use of this technology with arrays of thousands of nanopores will contribute to the development of transcription factor binding inhibitors

    Representation of electrostatic surface potentials.

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    <p>The electrostatic potentials are calculated on the X-ray structures of 14-3-3γ (PDB 2B05) (A, C, E) and 14-3-3ζ (PDB 2O02) (B, D, F). The electrostatic potentials are visualized on the solvent accessible surface in panel A–D with values colored from blue to red (from +2 kT/e to −2 kT/e). Panels E and F show the iso-contours of the electrostatic potential (+1 kT/e to −1 kT/e). The area with stronger positive electrostatic potential in 14-3-3γ (A) compared with 14-3-3ζ (B) corresponds to residues in helices A, B and D in the dimerization region that involves the N-terminal from each subunit (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049671#pone.0049671.s001" target="_blank">Figure S1</a>). Positive areas are predicted to contact the membrane in an initial phase driven by the electrostatics, while the negative areas on the convex side of the protein are possibly involved in tuning the orientation of the positive dimerization region towards the membrane. The bound phosphopeptides were omitted prior to the calculations of the electrostatic potentials.</p

    MD simulations of phosphopeptide-bound (holo-14-3-3γ) and ligand-free (apo-14-3-3) structures.

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    <p>A) Structural deviation from the initial X-ray structure (PDB 2B05) along the simulation time (100 ns) for phosphopeptide (RAIpSLP)-free apo (green and red lines, one line for each monomer A and B) and phosphopeptide-bound holo (blue and black lines for each monomer); RMSD, root mean square deviation. B) Positional fluctuations (root mean square fluctuations (RMSF)) along the holo (black bars) and apo (blue lines) 14-3-3γ. Helices are indicated schematically as black stripes. C) Backbone ribbon overlay representation of the dimeric structures obtained at the end of the MD simulations (100 ns) of holo (blue) and apo (green) 14-3-3γ. The inset (D) shows the residues mutated in this study (Tyr117, His158, His164 and His195) in stick representation, and the averaged distance between His158 and His195 at the end of the simulations. E, F) Representation of surface electrostatic potentials, calculated on the solvent accessible surface of the MD simulated structures of holo (E) and apo (F) 14-3-3γ, colored from blue to red (from +2 kT/e to −2 kT/e).</p

    Effect of mutation of specific histidine residues and of pH on the binding of 14-3-3γ to liposomes of PC∶PBPS (1∶1) measured by surface plasmon resonance (SPR).

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    <p>A) Representative sensorgrams for the binding of H158F- (⋅⋅⋅⋅⋅⋅⋅⋅), H195S- (–⋅⋅–⋅⋅–) and H158F/H195S- (– – –), comparative to wt-14-3-3γ (<sup>_______</sup>). The sensorgrams for H164E- and Y117F-14-3-3γ were similar to that of wt-14-3-3γ. The protein samples were prepared in 100 mM Na-phosphate, pH 7.4. B) Representative sensorgrams for the binding of wt-14-3-3γ as isolated, and diluted in either 100 mM Na-phosphate, pH 6.0 (<sup>_______</sup>), 100 mM Na-phosphate, pH 7.0 (⋅⋅⋅⋅⋅⋅⋅⋅), or 100 mM Tris-HCl, pH 8.0 (– – –). For both (A) and (B), liposomes were immobilized on the sensor chip at 4000–6000 RU, and each protein was applied at ∼50 µM subunit and injected at 25°C. The sample preparation buffers were used as running buffer.</p

    Effect of phosphopeptide ligand and kosmotropic salts on the binding of 14-3-3γ to negatively charged membranes.

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    <p>A) Representative sensorgrams for the binding of 14-3-3γ as purified (10 µM subunit) prepared in HBS-N buffer (10 mM Na-Hepes, pH 7.4, 150 mM NaCl) to liposomes made of PC∶PBPS (⋅⋅⋅⋅⋅⋅⋅⋅), or PC (– – –), and for the binding of 14-3-3γ (10 µM subunit) with THp-(1-43) (20 µM) in HBS-N buffer to liposomes made of PC∶PBPS (–⋅⋅–⋅⋅–) or PC (<sup>_______</sup>). B) The dependence of SPR responses (difference of RUs for binding to liposomes of PC∶PBPS and PC) on the subunit concentration of 14-3-3γ, alone (•) or with 2-fold concentration of THp-(1-43) (○). The binding isotherms were fitted using a single-rectangle, two-parameter equation providing S<sub>0.5</sub> values of 2.0±0.2 µM for the protein alone and 1.2±0.2 µM for the protein-phosphopeptide complex. C) Sensorgram for the binding to liposomes of PC∶PBPS of d14-3-3γ (extensively dialyzed in 10 mM Na-Hepes, pH 7.4) and further diluted in HBS-N buffer, alone (<sup>_______</sup>) or with a 2-fold concentration of THp-(1-43) (–⋅⋅–⋅⋅–) (running buffer HBS-N in both cases), and with 50 mM Na-phosphate, pH 7.4 (running buffer HBS-N with 50 mM Na-phosphate) (⋅⋅⋅⋅⋅⋅⋅⋅) or 50 mM Na-sulphate, pH 7.4 (running buffer HBS-N with 50 mM Na-sulphate) (– – –).</p
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