Schematic Representation of the WISE Strategy

<p>Schematic Representation of the WISE Strategy</p

Inferred Protein Interaction Networks

<div><p>(A) Protein interaction network mediated by the SH3 domains of the proteins characterized in this study. The SH3-containing proteins are represented as blue dots, while the prey partner proteins are represented as black dots. The interactions mediated by each SH3 are represented in a different color, and the edge thicknesses are proportional to the BLU intensity of the corresponding interaction, according to the scale described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020014#pbio-0020014-g003" target="_blank">Figure 3</a>.</p> <p>(B) The graph represents the interaction network mediated by the SH3 domains of Rvs167, Ysc84, Yfr024c, Abp1, Myo5, Sho1, Boi1, and Boi2 as determined by the two-hybrid approach (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020014#pbio-0020014-Tong1" target="_blank">Tong et al. 2002</a>). The interactions (edges) that were confirmed by our WISE method (BLU value higher than 25K) are colored in red or magenta. The interactions in magenta, differently from the ones in red, were not correctly inferred by the phage display approach. The interaction in orange was inferred by the phage display approach, but not confirmed by the WISE method. The network was visualized by the Pajek package (<a href="http://vlado.fmf.uni-lj.si/pub/networks/pajek/" target="_blank">http://vlado.fmf.uni-lj.si/pub/networks/pajek/</a>).</p></div

Comparison of the Phage Display Prediction and the Results of the SPOT Binding Test by the WISE Approach

<p>The quantitative results of the experiments in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020014#pbio-0020014-g001" target="_blank">Figure 1</a> are visualized with a graphical representation obtained with the tool EPCLUST available at <a href="http://ep.ebi.ac.uk/EP/EPCLUST" target="_blank">http://ep.ebi.ac.uk/EP/EPCLUST</a>. The PepSpot data, represented in red in a semiquantitative scale, is compared to the phage display prediction. Only peptides with BLUs (measured on a Lumi-Imager^TM) higher than 25K are included in the representation. The red intensity scale corresponds to BLU values in the ranges 25K–35K, 35K–45K, 45K–55K, 55K–85K, and larger than 85K, where higher BLU values correspond to a brighter red. Peptides that obtained a high score with the phage display-derived position-specific scoring matrix (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020014#pbio-0020014-Tong1" target="_blank">Tong et al. 2002</a>) are in brighter green. Peptides with a lower score are represented with a correspondingly lighter green according to an arbitrary linear scale.</p

Scanning of the Human Proteome in Search of Ligands for the Amphiphysin and Endophilin SH3 Domains

<p>The relaxed target peptide consensi (right) were derived from the available phage display experimental data and used to search the human proteins contained in the SwissProt/TREMBL database with the software ScanProsite, found at <a href="http://us.expasy.org/tools/scanprosite/" target="_blank">http://us.expasy.org/tools/scanprosite/</a>.</p

WISE Screening of the Binding Potential of Yeast SH3 Domains

<p>Seven GST–SH3 domain fusion proteins were challenged with peptides that match different relaxed consensi: class 1 (R/K)xxPxxP and class 2 PxxPx(R/K) . The Myo5 SH3 domain was also tested with peptides matching (F/P/L/W/A/E)xx(W/Y/L/M/F/H)xxPxxP, while the Abp1 membrane contains peptides matching either xxPx(K/R)P or Pxxx(K/R)P. In the design of these relaxed patterns, we first aimed at defining regular expressions that could retrieve from the proteome all the peptides that had been demonstrated, to bind to the domain under consideration. Whenever the number of matching peptides did not exceed an arbitrary chosen threshold of 1,500, we used subjective considerations about sidechain similarities to further relax the search pattern. The three spots near the membrane corners contain peptides that bind to the anti-GST antibody. The intensity of these spots was used for normalization.</p