4 research outputs found
Further testing of the polycation-Ο model.
<p>Designed mutant EADs (left) were tested for transcriptional activity and simulated binding. Full peptide sequences are given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003239#pcbi.1003239.s001" target="_blank">Fig. S1</a>. Y residues for all peptides are shown in magenta as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003239#pcbi-1003239-g001" target="_blank">Fig. 1</a> and the key residues are similarly depicted. Protein expression levels were determined by Western blot analysis of epitope-tagged activator proteins in extracts from transfected cells using KT3 antibody (right). The histograms show percentage experimental activities (black) and simulated (grey) relative to that of the first sequence (100%) in each experiment. Estimated errors for simulated are standard deviations from ten independent simulations. (A) Efficacy of different aromatic moieties. All Ys in 5Yn (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003239#pcbi-1003239-g001" target="_blank">Fig. 1A</a>) were replaced by W (yellow) or F (orange). The variation of well depth for cation-F (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003239#pcbi-1003239-g001" target="_blank">Fig. 1B</a>) entails a range of relative from 24% to 80% and the latter is plotted here. (B) Effect of adding anions (Asp, shown in blue). (C) Effect of adding cations (Arg, shown in green).</p
IDP-target binding in the analytical model.
<p>To match the chain simulation model, we used β=β438.0 Γ
<sup>3</sup>, where <i>b</i>β=β3.8 Γ
is the β virtual bond length and β=β6 Γ
is the capture radius for a cation-Ο contact in the chain model. (A) The IDP's chain length <i>n</i>β=β66, with <i>k</i>β=β6 (corresponding to the sequences in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003239#pcbi-1003239-g001" target="_blank">Fig. 1</a>). was computed for different values. β=β32 for the target and <i>V</i>β=β(600 Γ
)<sup>3</sup> as in the simulations [hence β=β13.1]. Inset: The energy () and entropy () components of for β=ββ3.5. Results in (BβD) are also for β=ββ3.5. (B) Effects of <i>k</i> and <i>V</i> on binding; β=β32; β=β1/(600 Γ
)<sup>3</sup> is used as a reference IDP concentration. The black curves show at for hypothetical sequences with <i>k</i>β=β9, 8, 7, 6, 5, 4, and 3 (from top to bottom), <i>n</i>β=β66 for <i>k</i>β€6 and <i>n</i>β=β for <i>k</i>β₯7. The blue curves are for the <i>k</i>β=β6 sequences for three IDP concentrations with <i>C</i>β=β0.25, 3.0, and 10.0 (from top to bottom). (C) for <i>k</i>β=β6 sequences at <i>C</i>β=β1 on different targets of the same size with different β=β8, 16, 32, 48, 64, and 80 (from left to right; see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003239#pcbi.1003239.s012" target="_blank">Text S1</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003239#pcbi.1003239.s005" target="_blank">Fig. S5B</a>). (D) of the <i>k</i>β=β6 sequences at different IDP concentrations <i>C</i>β=β10.0, 5.0, 4.0, 3.0, 2.0, 1.0, 0.5, 0.33, and 0.25 (from top to bottom).</p
Model for molecular recognition by EAD.
<p>The EAD peptide is depicted here as a string of beads with aromatic (Y) residues in magenta and other residues in grey (see also <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003239#pcbi.1003239.s002" target="_blank">Fig. S2</a>). The target protein (Target) is generic and the number/distribution of surface positively charged (R) residues for real targets are unknown. Rs are chosen over Ks simply because Rs are more commonly paired with Ys in cation-Ο interactions. Binding is driven predominantly by cation-Ο interactions between Ys and Rs. A key postulate of the model is that the EAD remains disordered irrespective of binding and exists as a dynamic ensemble. Two general, high-probability states are depicted: (A) At low Y number the probability of EAD rebinding is low; dissociation is favored. (B) At higher Y number the probability of rebinding is sufficient to counteract dissociation and maintain binding.</p
Effect of Y density and distribution on EAD activity.
<p>(A, B) The EAD peptides (left) were tested for relative transactivation (black) and simulated (grey), shown in the same style as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003239#pcbi-1003239-g003" target="_blank">Fig. 3</a>. (A) 7Yn (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003239#pcbi-1003239-g001" target="_blank">Fig. 1A</a>) with Y density denoted normal (n or <i>k</i>β=β6) was compared with 7Yn/2 (Y density βΌ1/2 of 7Yn, <i>k</i>β=β12) and 7Yn/4 (Y density βΌ1/4 that of 7Yn, <i>k</i>β=β24). The actual simulated for 7Yn is 0.11. (B) 10Yn (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003239#pcbi-1003239-g001" target="_blank">Fig. 1A</a>; <i>k</i>β=β6) was compared with 5Y (<i>k</i>β=β12) and the sequence 5YP which has 5 pairs of sequentially adjacent Ys. The asterisk indicates that 5Y activity is overstated due to relatively higher expression of 5Y protein. (C) Analysis using our analytical model. All were for β=β32, <i>C</i>β=β1, and β=ββ3.5 except the data point plotted as open circle (β=ββ2.6) was for β=ββ5.1. The solid line shows results for <i>k</i>β=β6 and <i>n</i>β=β66. The upper and lower dashed lines provide results for <i>k</i>β=β12 with chain lengths <i>n</i>β=β66 and <i>n</i>β=β71 respectively. The diamonds show results (from bottom to top) for 7Yn, 7Yn/2, and 7Yn/4 in (A), which have chain lengths <i>n</i>β=β66, 86, and 156 respectively. To facilitate comparison with the β=β7 data in (A), β=β7 is marked by the vertical dotted line. The squares show results for 5Y (β=β5; β=β4.0) and 10Yn (β=β10; β=ββ3.2) in (B), both with <i>n</i>β=β66. As discussed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003239#pcbi.1003239.s012" target="_blank">Text S1</a>, the model represented by the open circle may be applied to 5YP in (B) with β5.1 as the interaction energy between a cation and two adjacent aromatic residues.</p