Comparison of the two WT permutation patterns.

<p>Intensities were normalized with respect to the mean intensity recorded for the scFv1F4—WT peptide interaction. Black and grey bars correspond to the normalized intensities in arrays A and B, respectively. White bars represent the WT sequence (normalized value = 1). Stars indicate single replacements in starting variants (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143374#pone.0143374.t001" target="_blank">Table 1</a>).</p

Normalized SPR kinetic curves.

<p>The purified scFv1F4 (10 nM in HBS) was injected over peptide surfaces. The WT curve is shown in red, single variants in light grey, double variants in dark grey and the triple variant in black. The curves were superimposed using the software TraceDrawer (Ridgeview Instruments AB, Uppsala, Sweden).</p

Binding parameters deduced from SPR measurements in HBS.

<p>^a Sequence replacements relative to WT are in bold and underlined.</p><p>Binding parameters deduced from SPR measurements in HBS.</p

Spot fluorescence data for the starting peptides.

<p>^a Sequence replacements relative to WT are in red.</p><p>^b Mean intensities > 10% that of the WT peptide are in bold.</p><p>^c Standard deviation calculated as <math><mrow><mi>s</mi><mo>=</mo><msqrt><mrow><mn>1</mn><mrow><mi>N</mi><mo>−</mo><mn>1</mn></mrow><mo>∑</mo><mrow><mi>i</mi><mo>=</mo><mn>1</mn></mrow><mi>N</mi><mrow><mrow><mrow><mo>(</mo><mrow><msub><mi>x</mi><mi>i</mi></msub><mo>−</mo><mi>x</mi><mo>¯</mo></mrow><mo>)</mo></mrow></mrow><mn>2</mn></mrow></mrow></msqrt></mrow></math></p><p>^d The permutation of the WT decapeptide in array A was described in Vernet <i>et al</i>., 2015 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143374#pone.0143374.ref028" target="_blank">28</a>].</p><p>Spot fluorescence data for the starting peptides.</p

Context dependence of the effect on k_d of replacements.

<p>Positions 11, 12 and 13 in the peptide sequences are shown in red, blue and green, respectively. The filling is white for the WT residue (D₁₁, P₁₂, Q₁₃) and colored for the modified residue (S₁₁, V₁₂, F₁₃). The ratio k_d variant / k_d WT, as measured by SPR in HBS (black) or HBS200 (grey), is given next to each arrow, together with the nature of the replacement.</p

Stained PEPperCHIP® array B.

<p>The permutation scans of the WT peptide (upper left) and of variant ⁶TAMFQDP<b>F</b>ER¹⁵ are highlighted by white frames. Replaced positions together with the WT amino-acid are indicated on the left of the upper left frame. Amino-acids introduced at each position are indicated on top of the frame. Duplicated spots are easily identified.</p

Correlation between SPR constants and fluorescence signals.

<p>The k_d (A, B) and ΔG (C, D) values are plotted against fluorescence signals from arrays A (A,C) and B (B,D). The k_d as measured in HBS (grey markers) and HBS-200 (white markers) is shown in log scale. Peptide names are indicated in A and B.</p

Permutation patterns for variants modified at positions 11, 12 and 13.

<p>Variant patterns are represented as red lines and superimposed with the WT pattern (blue). Fluorescence signals were normalized with respect to that recorded with each starting peptide. Patterns at the replaced positions are in green for double variants and not shown for single variants.</p

HPV E6-mediated p53 degradation.

<p>A) HA-tagged p53 levels were visualized by Western blot after co-transfection with HA-tagged HPV E6 from HPV11, HPV16 and HPV18 into C-33A cells. Lanes 1, 3, 5 and 7 show results from co-transfection of HA-p53 and vectors indicated at the top of the figure. Lanes 2, 4, 6 and 8 show the p53 levels after treatment with MG132, as indicated at the top of the figure. β-tubulin was visualized as a loading control. (B) Half life of HA-p53 in transfected 293T cells. 293T cells were transfected with HA-p53 and control (pQCXIN) or E6 ORFs (HPV11, HPV18, HPV53, HPV56 and HPV66). The band intensities were determined from the scanned Western blot using ImageQuant and the signals at time 0 were defined as 100%. The band intensities of the indicated time points were normalized to time 0.</p

Alignment of HPV alpha E6 ORFs.

<p>The alignment of all 27 E6 ORFs tested in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone-0012816-g002" target="_blank">Figure 2</a> is shown. HPV types degrading p53 are shown at the top of the alignment and those not degrading p53 are shown at the bottom. The shaded region represents a proposed E6-AP binding domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Liu1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Pim1" target="_blank">[26]</a>. The amino acid sequences of the E6 ORFs were aligned using Clustal X (version 1.81) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012816#pone.0012816-Thompson1" target="_blank">[36]</a>. The amino acid at position 31 (arrow, <b>bold</b>) was associated with p53 degradation (p<0.01). “_” indicate gaps, whereas “.” indicates identical residues with the HPV16 E6 amino acid sequence shown in the top row.</p