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

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

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

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

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

Chemical Library Screening Using a SPR-Based Inhibition in Solution Assay: Simulations and Experimental Validation

We have developed a surface plasmon resonance (SPR)-based inhibition in solution assay (ISA) to search for inhibitors of the medium affinity (<i>K</i>_D = 0.8 μM) interaction between an E6-derived peptide (E6_peptide) immobilized on the sensor and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are widespread protein-protein interaction modules that recognize the C-terminus of various partners. Simulations indicated that relatively low compound concentrations (10 μM) and limited peptide densities (<i>R</i>_max < 200 resonance units) should allow the detection of inhibitors with a target affinity close to 100 μM, which was then demonstrated experimentally. ISA screening, carried out on the Prestwick Chemical Library® (1120 compounds), identified 36 compounds that inhibited the interaction by more than 5%. Concentration-dependent ISA, carried out on a subset of 19 potential inhibitors, indicated that 13 of these indeed affected the interaction between MAGI-1 PDZ1 and the E6_peptide. No effect was observed for 84 compounds randomly chosen among noninhibitors. One of the four best inhibitors was a peptide binder, and three were PDZ binders with <i>K</i>_D in the 10–50 μM range. We propose that a medium (μM) affinity between the target and surface-bound partner is optimal for SPR-based ISA screening

Effect of successive deletion in <i>rpsA</i> performed at the original <i>rpsA</i> locus on cell growth.

<p>(A) Growth was compared between WT strains (Wt, <i>rpsA1</i>) and strains carrying deletions of domains 6 (Δ6) and of domains 5 and 6 (Δ56) in <i>rpsA</i> on LB plates at 37°C. The <i>E. coli</i> strains are AnK02 (WT), MS77 (<i>rpsA1</i>), MS78 (Δ6), and MS79 (Δ56). (B) Measurements of the doubling times of various strains. The growth was done in LB medium at 37°C. The strains were identical to those of the panel A. (C) The growth was compared in strains carrying deletions of domains 5 and 6 (Δ56), 4 to 6 (Δ4–6), 3 to 6 (Δ3–6), 2 to 6 (Δ2–6) in <i>rpsA</i>. They were complemented with the plasmid pNK34, which carries WT <i>rpsA</i> under the control of the hybrid <i>trc</i> promoter with the <i>lac</i> operator. The experiments were done in the presence of IPTG (+IPTG) or in the absence of IPTG (−IPTG). Strains are MS79pNK34 (Δ56), MS84pNK34 (Δ4–6), MS83pNK34 (Δ3–6), and MS82pNK34 (Δ2–6).</p