Interaction of the fluorescent extrinsic probe bis-ANS with NS3 at pH 6.4 and 7.2.

<p>bis-ANS concentrations ranging from 0 to 8 µM were used to compare NS3hel (A) and NS3FL (B) hydrophobic clefts exposure at pH 6.4 (closed circles) and 7.2 (open circles). The inset in the graph A shows a reduction in the y-axis scale to demonstrate more clearly the effect of increasing bis-ANS fluorescence at both pH. Each point corresponds to the mean of the normalized bis-ANS fluorescence intensity obtained in three independent experiments. Spectra were acquired at 25°C in buffer solutions composed of 50 mM MOPS-NaOH (pH 6.4 or 7.2), 200 mM NaCl, 5 mM β-mercaptoethanol and 5% glycerol. The protein concentration was 1 µM.</p

Analysis of the pH effects on the NS3 secondary structure upon chemical denaturation.

<p>A) CD spectra of 10 µM NS3hel acquired at pH 7.2 in the absence of Gdn.HCl (solid line), or presence of 2.5 M (dashed line) and 5 M (dotted line) Gdn.HCl. The spectra were the average of three scans after subtracting the buffer baselines. Each spectrum was converted into molar ellipticity using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115941#pone.0115941.e004" target="_blank">Equation 4</a> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115941#s4" target="_blank">Material and Methods</a>). B) The ellipticity values at 222 nm (θ₂₂₂) at each Gdn.HCl concentration (from 0 to 5 M) were used to compare the secondary structure stability of NS3hel at pH 6.4 and 7.2 and to calculate the degree of denaturation using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115941#pone.0115941.e005" target="_blank">Equation 5</a> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115941#s4" target="_blank">Material and Methods</a>). Closed (pH 6.4) and open circles (pH 7.2) represent the degree of denaturation at each Gdn.HCl concentration. Spectra were acquired at 25°C in buffer solutions composed of 50 mM MOPS-NaOH (pH 6.4 or 7.2), 200 mM NaCl, 5 mM β-mercaptoethanol and 5% glycerol. The protein concentration was 10 µM.</p

Effects of pH on NS3 binding to ssDNA.

<p>Increasing protein concentrations (from 0 to 10 µM NS3hel and 0 to 5 µM NS3FL) were used to compare NS3hel (A) and NS3FL (B) binding to a fluorescently-labeled ssDNA at pH 6.4 and 7.2 and to calculate the dissociation constants (K_d) between ssDNA and the constructs. Closed (pH 6.4) and open circles (pH 7.2) represent the mean of fluorescence anisotropy obtained in three independent experiments. Data were obtained at 25°C and assay buffers contained 25 mM MOPS-NaOH (pH 6.4 or 7.2), 2 mM MgCl₂ and 25 nM of the fluorescently labeled ssDNA.</p

Effects of ssDNA binding on NS3 structure monitored by Trp fluorescence quenching and bis-ANS binding.

<p>Increasing ssDNA concentrations (from 0 to 1 µM) were used to compare the Trp fluorescence quenching of NS3hel and NS3FL (A and B, respectively) and the bis-ANS binding to these proteins (C and D, respectively) at pH 6.4 (closed circles) and 7.2 (open circles). Each point represents the mean of Trp fluorescence quenching or bis-ANS binding obtained in three independent experiments. Spectra were obtained at 25°C and assay buffers contained 25 mM MOPS-NaOH (pH 6.4 or 7.2), 2 mM MgCl₂ and 1 µM of purified proteins.</p

Fluorescence quenching of NS3 Trp residues by acrylamide at pH 6.4 and 7.2.

<p>Acrylamide concentrations ranging from 0 to 156 mM were used to monitor the exposure of the Trp residues of NS3hel (A) and NS3FL (B) at pH 6.4 (closed circles) and 7.2 (open circles) and to calculate the Stern-Volmer constant (K_sv) using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115941#pone.0115941.e003" target="_blank">Equation 3</a> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115941#s4" target="_blank">Material and Methods</a>). Each point corresponds to the mean of tryptophan fluorescence quenching by acrylamide obtained in three independent experiments. Spectra were acquired at 25°C in buffer solutions composed of 50 mM MOPS-NaOH (pH 6.4 or 7.2), 200 mM NaCl, 5 mM β-mercaptoethanol and 5% glycerol. The protein concentration was 1 µM.</p

Effects of bis-ANS binding on ATPase activity at different pHs.

<p>Increasing bis-ANS concentrations (from 0 to 300 µM) were used to compare the effect of bis-ANS binding on the ATPase activity of NS3hel (A) and NS3FL (B). Closed (pH 6.4) and open circles (pH 7.2) represent the mean of the residual ATPase activity obtained in three independent experiments and bars indicate the standard error. Reactions were performed at 30°C during 60 min using 40 mM MES-KOH (pH 6.4) or Tris-HCl (pH 7.2), 5 mM DTT, 5 mM MgCl₂, 100 mM KCl, 1 mM ATP and 0.1 µM of purified proteins. IC₅₀ values were calculated using <i>Sigma plot</i> ver. 10.0 after plotting the dose-response curve of bis-ANS concentration versus residual ATPase activity.</p