Fig 3. Template-free de novo design strategy.
(A) Protein topologies that are compatible with each motif are enumerated in the 2D space. Selected topologies are then projected into the 3D space using idealized SSEs, and their relative orientation is sampled parametrically. Distance constraints are derived from selected topologies to guide in silico folding and sequence design using Rosetta. (B) Designed sequences were screened for high-affinity binding and resistance to chymotrypsin to select stably folded proteins, as revealed by next-generation sequencing (NGS). (C) For the S4_2 design series, enrichment analysis revealed a strong preference for one of the designed helical orientations (S4_2_bb2, green) to resist protease digestion and to bind with high affinity to 101F. (D) To ensure epitope integrity, S0_2_bb3 was screened for binding to both D25 and 5C4. Sequences highly enriched for both D25 and 5C4 binding show convergent sequence features in the critical core position 28 of the site 0 scaffold. (E-F) Thermal melting curves measured by CD for best designs (S4_2.45 (E) and S0_2.126 (F)) showing high thermostability. (G-H) Dissociation constants (KD) of S4_2.45 to 101F (G) and S0_2.126 to D25 (H) as measured by SPR. E: enrichment.