LIP and the lowest stability foldon in Cytochrome c.

<p>(A) Polarity ratio and packing density profiles of Cytochrome c (PDB: 1HRC, Resolution  = 1.9 Å). The segment shadowed in grey corresponds to the lowest stability region of the protein (infrared foldon: residues 40–57) according to equilibrium and kinetic H-exchange NMR experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048212#pone.0048212-Krishna1" target="_blank">[39]</a>. The light blue bar indicates the only LIP in Cytochrome c, which includes residues 40–45 and is located in the unstable foldon. (B) Ribbons representation showing the unstable foldon in grey. In the charts, the polarity and packing cutoffs are indicated as grey dashed lines</p

LIPs in the late transition state ensemble of barnase folding.

<p>(A) Polarity ratio and packing density profiles of barnase (PDB: 1A2P, Resolution  = 1.5 Å). The segments shadowed in grey correspond to the regions displaying φ-values equal to or lower than 0.5 in the late transition state of barnase folding (TS2) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048212#pone.0048212-Salvatella1" target="_blank">[47]</a>. The light blue bars indicate the three LIPs in barnase: 20–30, 44–57 and 65–89. They closely correspond to the segments exhibiting low φ-values in the transition state (19–37, 39–55 y 72–88). (B) Ribbons representation showing in grey the transition state regions with low φ-values. In the charts, the polarity and packing cutoffs are indicated as grey dashed lines.</p

Identification and structural characterization of LIPs in apoflavodoxin.

<p><b>A</b>) Stacked-aligned profiles for polarity ratio and packing density in <i>Anabaena</i> PCC 71191 apoflavodoxin (PDB: 1FTG, Resolution  = 2.0 Å). The property values (see definitions in Materials and Methods) are plotted against the position of the fourth residue of an eight-residue probe fragment. The segments encompassing residues 87–108 and 118–152, which have been found to be unstructured in the equilibrium intermediate of this protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048212#pone.0048212-AyusoTejedor1" target="_blank">[31]</a>, are highlighted in grey. We also show in grey dashed lines the polarity and packing cutoffs. <b>B</b>) Surface representation of buried atoms at interfaces 87–107 (yellow) and 118–152 (red) and the associated interacting fragments (in cartoon representation) colored purple and blue, respectively. <b>C</b>) Surface representation of the buried atoms according to our characterization of polar light interfaces (LIPs). The LIPs 87–99 (yellow), 120–133 (red) and 140–155 (cyan) are shown and the associated interacting fragments are colored purple, blue and green and are depicted in cartoon representation.</p

LIPs and the unfolded domain of the α-Lactalbumine Molten globule.

<p>(A) Polarity ratio and packing density profiles of α-Lactalbumine (PDB: 1HML, Resolution  = 1.7 Å). The segment shadowed in grey corresponds to the β-domain (residues 40–81), the one that lacks secondary structure in the molten globule intermediate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048212#pone.0048212-Schulman1" target="_blank">[43]</a>. The light blue bars indicate the two LIPs in α-Lactalbumine, encompassing residues 35–51 and 64–70, and essentially defining the β-domain. (B) Ribbons representation showing the unstable β-domain in grey. In the charts, the polarity and packing cutoffs are indicated as grey dashed lines.</p

LIPs and the unfolded regions of the equilibrium (and kinetic) intermediate of Indole-3-glycerol phosphate synthase.

<p>(A) Polarity ratio and packing density profiles of Indole-3-glycerol phosphate synthase (PDB: 2C3Z, Resolution  = 2.8 Å). The segments shadowed in grey correspond to the unfolded regions of the equilibrium intermediate of chemical unfolding (intermediate Ia), which coincides with the on-pathway kinetic folding intermediate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048212#pone.0048212-Gu1" target="_blank">[44]</a>. The light blue bars indicate the five LIPs in Indole-3-glycerol phosphate synthase. LIPs 7-18 and 23–40 map onto the N-terminal unfolded region of the protein (1–47). The next LIP, 58–68, defines the loop that is unfolded even in the native state (59–68). Finally, LIPs 148–170 and 178–205 are located at the C-terminal unfolded segment of the protein (162–220). (B) Ribbons representation showing the unfolded regions of the intermediate in grey. In the charts, the polarity and packing cutoffs are indicated as grey dashed lines</p

Dissociation constants for selected compounds binding to drug-resistance-associated NS3 protease variants (25°C, 100 mM sodium acetate, 2 mM EDTA, pH 5).

a<p>The inactive S139A mutant represents the pseudo-wild-type, behaving similarly to wild-type (WT) NS3 protease in terms of structural stability, substrate binding affinity and NS4A activation.</p><p>Relative error in the parameters is 15%.</p

In vitro enzymatic inhibition of NS3 protease.

<p>(Left panel) Fluorescence intensity measured as a function of time (wavelengths of 380 nm and 500 nm for excitation and emission, respectively) for the substrate catalysis by NS3 protease in the absence (open squares) or presence of compound 1 at 25 µM (closed squares), in sodium acetate pH 5, 0.8 nM free Zn⁺² concentration. (Right panel) NS3 protease activity was determined as the initial slope of the curves. The percentage of activity is calculated as the quotient between the activity of NS3 protease in the presence (25 µM) and the absence of a given compound (compound 1, closed squares; compound 5, open squares).</p

Inhibition of HCV replicon in cell assays.

<p>Evaluation of potency and cytotoxicity of the selected compounds in cell assays. HCV replicon replication rate (white bars) and cell survival (closed squares) were assessed in cell culture at increasing compound concentration to determine EC50 and CC50. For compounds 1 and 3 assays with lower concentrations were performed in order to reliably determine the EC50. *UTC: untreated controls.</p

Experimental screening for ligands binding to the Zn⁺²-free NS3 protease.

<p>Thermal denaturation curves of Zn⁺²-free NS3 protease followed by ANS fluorescence in the presence of different compounds (100 mM sodium acetate, pH 5, 2 mM EDTA). Inset: Thermal denaturation of Zn⁺²-free NS3 protease followed by tryptophan intrinsic fluorescence (average energy of spectra). The unfolding transition restricted to the temperature range accessible in the microplate fluorescence reader for the library screening is indicated in closed squares for comparison.</p

Action mechanism of the allosteric inhibitors.

<p>The proper folding of the NS3 protease towards the active conformation, promoted <i>in vivo</i> by its two cofactors (Zn⁺² and NS4A), is prevented by the compounds through stabilization of the inactive partially folded Zn⁺²-free conformation.</p