Model of the apoA-I peptide probably involved in the heparin binding site.

<p>(A) A model was built by using the Swiss-model database (<a href="http://www.expasy.org/" target="_blank">www.expasy.org</a>). The sequence loaded involves residues 143–186 in the wt form (PLGEEMRDRARAHVDALRTHLAPYSDELRQ<b>R</b>LAARLEALKENGG). Ball-and-stick representation was obtained with the Avogadro’s software. Positively charged residues are as follows: Arg are in green, His, in yellow and Lys, in pink. (B) Same for the Arg173Pro variant (PLGEEMRDRARAHVDALRTHLAPYSDELRQ<b>P</b>LAARLEALKENGG). The Pro residue incorporated with the mutation is shown in light blue.</p

Protein binding to SDS.

<p>A) The wt and Arg173Pro variants (0.2 mg/mL) were incubated (either at pH 7.4 or 5.0) in the absence or in the presence of 0.2 mM SDS. After 48 h at 37°C ThT was added to a 1:1 molar ratio to protein. Fluorescence intensity was quantified in the microplate reader at 480 nm (excitation set at 430 nm). Statistically significant differences between experimental conditions were evaluated by ANOVA followed by Tukey’s test. Bars correspond to means ± SE. The symbol * denotes difference with respect to wt at p<0.01. B) The effect of salt concentration was tested by incubating Arg173Pro (0.2 mg/mL) at pH 7.4 in the absence or the presence of 0.2 mM SDS plus 0, 100 and 400 mM ClNa. Incubation was performed as in A). The symbol # denotes difference with respect to the protein in the absence of ClNa at p<0.01</p

Characterization of heparin binding to apo A-I variants.

<p>Polyacrylamide gradient gel electrophoresis (PAGGE, 4–25%) under native conditions. wt (lane 1) and Arg173Pro variants (lane 2); lanes 3 and 4 correspond to wt and Arg173Pro variants plus heparin (added at a 2:1 heparin to protein molar ratio), after 48 h incubation at 37°C at pH 7.4. B) Characterization of the morphology of Arg173Pro aggre<b>g</b>ates with he<b>p</b>arin. Analysis of images observed under AFM. Proteins (0.5 mg/mL) were incubated for 24 h and loaded onto mica plates. The bar shows the scale indicated in the image. C) Distribution of the height of oligomers resulting from the measurement in the <i>z</i>-plane. D) Proteins (0.2 mg/mL in citrates phosphates McIlvaine’s buffer, pH 5.0) were incubated for 48 h at 37°C in the presence or in the absence of heparin at a 1:1 molar ratio. Binding of ThT was measured as described above. Bars correspond to means ± SE. Statistically significant differences between experimental conditions were evaluated by Student t-test. Bars correspond to means ± SE. The symbol * denotes the difference with the same protein without heparin at p<0.01.</p

Characterization of Arg173Pro aggregates at pH 7.4.

<p>A) Binding of ThT to apoA-I. Proteins (0.2 mg/mL in citrates phosphates McIlvaine’s buffer, pH 7.4) were incubated for 48 h at 37°C and ThT added to each sample at a 1:1 molar ratio to protein. Fluorescence was quantified in the microplate reader at 480 nm (excitation set at 430 nm). Bars correspond to means ± SE. Statistically significant differences between experimental conditions were evaluated by Student t test. Symbol * denotes a difference with respect to wt at p<0.05. B) Characterization of the morphology of Arg173Pro aggregates after analysis of AFM images. Protein (0.5 mg/mL) was incubated for 24 h and loaded onto mica plates. Heterogeneous size oligomers covering the surface of the mica were predominant in the sample. The inset represents a frame at higher magnification, allowing us to observe elongated aggregates with a protofibrillar shape. Bars show the scale used in each case. C) Histogram distribution of the height of the oligomers obtained from measurements in the <i>z</i>-plane.</p

Fluorescence characterization of wt and Arg173Pro forms at pH 7.4 and 5.0.

<p>^a Stern-Volmer constant for the quenching of Trp residues by acrylamide.</p><p>^b Free energy change of unfolding and</p><p>^c concentration at which half of the protein is unfolded, respectively, calculated from equilibrium unfolding curves, as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124946#pone.0124946.ref018" target="_blank">18</a>] and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124946#pone.0124946.g002" target="_blank">Fig 2</a>. N/A, not assayed</p><p>^d Light scattering of proteins (0.2 mg/mL) measured after 48 h incubation at 37°C with excitation and emission wavelengths set at 350 nm.</p><p>^e Fluorescence resonance energy transfer efficiency (E), calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124946#pone.0124946.e002" target="_blank">Eq (2)</a>. F_DA and F_D are the intensity of Trp emission in the presence and in the absence of the acceptor molecule bis-ANS (excitation set at 295 nm and emission at 335 nm).</p><p>^f Intensity ratio of the fluorescence associated to bis-ANS and Trp in the samples described above, measured by direct excitation of each dye, as described in ‘Methods’.</p><p>Fluorescence characterization of wt and Arg173Pro forms at pH 7.4 and 5.0.</p

Spectroscopical characterization of protein structure.

<p>Wt and Arg173Pro variants (0.1 mg/mL) were dissolved in citrates phosphates McIlvaine’s buffer, pH 7.4. A) Far-UV circular dichroism. Dark and light grey lines correspond to wt and Arg173Pro proteins, respectively. B) Proteins were incubated with increasing concentrations of bis-ANS. The probe was excited at 360 nm, and the emission recorded at the wavelength of maximum fluorescence for this probe (488 nm). Filled and open circles represent the experimental data points for wt and Arg173Pro, respectively.</p

Protein structure, stability and aggregation tendency at pH 5.0.

<p>A) Proteins were dissolved in citrates phosphates McIlvaine’s buffer, pH 5.0 and far-UV circular dichroism measured as describe above. Dark and light grey solid lines correspond to the wt and Arg173Pro variants, respectively. To facilitate comparison, dashed lines correspond to both proteins measured at pH 7.4 (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124946#pone.0124946.g001" target="_blank">Fig 1A</a>); B) Arg173Pro (0.1 mg/ml, pH 5.0) was incubated with increasing concentrations of GndHCl and the center of mass of the Trp fluorescence measured as described above (filled circles). Here again, to facilitate comparison, dashed lines correspond to the protein measured at pH 7.4 (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124946#pone.0124946.g002" target="_blank">Fig 2</a>). C) Analysis of energy transfer from Trp to bis-ANS. The Arg173Pro variant was dissolved in citrates phosphates McIlvaine’s buffer (0.1 mg/mL) and Trp fluorescence measured (with excitation set at 295 nm, continuous line). Bis-ANS was added next and spectra measured (with excitation set at 295 nm, dotted line). Direct excitation of bis-ANS was achieved at 395 nm (dashed line). Dashed and dotted line represents bis-ANS excitation at 295 nm in protein-free buffer. Efficiency was calculated by measuring the intensity of Trp fluorescence at the wavelength of maximum emission (335 nm). D) Binding of ThT to apoA-I. Proteins (0.2 mg/mL in citrates phosphates McIlvaine’s buffer, pH 5.0) were incubated for 48 h at 37°C and ThT added to a 1:1 molar ratio to protein. Fluorescence intensity was quantified in the microplate reader at 480 nm (excitation set at 430 nm). Bars correspond to means ± SE. Statistically significant differences between experimental conditions were evaluated by Student t-test. The symbol * denotes a difference with respect to wt at p<0.05.</p

Chemical denaturation as followed by GndHCl titration.

<p>Proteins (0.1 mg/mL in citrates phosphates Mc Ilvaine’s buffer, pH 7.4) were incubated with increasing concentrations of GndHCl. Trp fluorescence emission spectra were obtained by excitation at 295 nm, and scanning the emission between 310 and 420 nm. With these data, the center of mass was calculated for each sample. Filled and open circles represent the experimental data for the wt and Arg173Pro variants, respectively.</p