Designing specific peptides using CLASSY. A) Specificity sweep scheme. A sequence (black) is sought that binds a target (red) but not several undesired partners (gray) or itself. Panels from left to right illustrate iterations of the CLASSY procedure, during which the specificity gap Δ is increased. B) and C) A specificity sweep with MafG as the target and all other human bZIP coiled coils (except MafK, in the same family as MafG) and the design homodimer as undesired complexes. The plot in B corresponds to the cartoon in A. Red dots, black bars and gray bars represent energies of the design•target, design•design, and design•other bZIP complexes, respectively. C plots design•target complex stability vs. specificity (Δ). Portions of several designed complexes are shown using helical wheels (orange highlights amino-acid changes from the previously shown sequence). The rightmost solution is anti-SMAF.

Properties of designed peptides compared to human bZIP leucine-zippers. A) Hierarchical clustering of interaction profiles for 33 human peptides and 48 designs; an interaction profile consists of the array signals for interactions with 33 surface-bound human peptides. Proteins on the surface are in columns and those in solution are in rows, with designed proteins and their interaction profiles in blue and human bZIP interaction profiles in yellow. B), C) Sequence logos for a, d, e, and g positions from the first 5 heptads of the 33 human bZIP leucine zippers in B) and the 48 designed peptides in C) (http://weblogo.berkeley.edu).

Experimental testing of anti-bZIP designs. A) Peptide array results for the most specific design identified for each human bZIP family. Columns show experiments using the indicated protein to probe an array. For the Specificity panel (left), designs in solution were used to probe human bZIPs and designs on the surface. In the Relative Stability panel (right), human bZIP targets were used to probe an array containing the cognate design of each target and 33 human bZIPs. Data are plotted as -log(F/F_max), with F the fluorescence signal on the array, such that the strongest interaction has a value of zero. Values of -log(F/F_max) above 1.0 were set to 1.0. Thick red circles – design•target; thin red circles – design interactions with siblings in the target family; grey squares – interactions with other human bZIPs; black squares – design•design. Designs are named using the family of their target. B) Solution testing of anti-SMAF complexes assayed using circular dichroism. In each panel, anti-SMAF alone is shown with dashed lines, the partner being tested with a solid line, the numerical average of these two signals with open circles (◦) and the mixture of the two peptides with closed circles (•). (B, C) Anti-SMAF interacts with target MafG (T_m ~ 38 °C). (D) Anti-SMAF interacts, at most, very weakly with cJun, the closest competitor according to microarray data. (E) There is no evidence for anti-SMAF interacting with MafB, a sequence closely related to the target. CD spectra in (B) were collected at 25 °C. Anti-SMAF unfolds with T_m ~12 °C. Similar data for other complexes are included in Supplemenatary Figures 3–8.