Figure 3.

ATF3 library a and e/g designs. During library design for PCA screening, peptide options were semirandomized. Lib_a: all five a positions were semirandomized to provide 12 options (FLIVYHNDSPTA⁵ = 248,832 options) while all 10 e/g positions were fixed as Q to provide a generic electrostatic option predicted to provide weak nonselective affinity. Lib_e/g: All 10 e/g positions were semirandomized to provide 3 options (QKE¹⁰ = 59,049 members) while core a positions were fixed as AIAIA to facilitate parallel dimeric coiled coil formation of low affinity to place a strong selection emphasis upon the electrostatic component. Shown are hydrophobic options at core interfacial positions (a) and charged/polar options which are present at flanking positions (e/g). In particular, the g and e positions have been semirandomized to provide attractive and repulsive options with corresponding positions on the target. Likewise, interfacial a positions were semirandomized to generate a range of options that included aliphatic hydrophobic side chains, as well as Asn, Ala, and aromatic options (Phe, Tyr, His). Positions c and d positions were fixed as A and L, respectively (position b2 was fixed as Y for quantification purposes). The appeal of the approach is that the two libraries can be exponentially combined to produce a library size of ∼14.7 billion members that is inaccessible to intracellular screening platforms.