Figure 1: Schematic overview of the Alphabody platform.

(a) Alphabodies are encoded by a single polypeptide featuring three α-helices (A–C) connected by linkers (L1, L2). The sequences of the three generic libraries (scLib_AC11, scLib_C9 and scLib_AC7) as well as the maturation library (matLib) used for biopanning are depicted. Amino-acid positions denoted with ‘x’ mark positions that were fully or partially randomized. The N terminus (N) of the Alphabody consists of the first 21 amino acids from the pelB leader sequence followed by an Asp in scLib_AC7 or Ser in all other cases. The linkers connecting the helices (L1 and L2) are identical within each library (La=T (GGSG)₄MS, Lb=T(GGSG)₄MD). The C terminus (C) differed between the generic libraries (TPGGSGGAAAHHHHHHGRAQ) and the maturation library (AAAHHHHHHQ) where in the latter the glutamine residue was encoded by an amber stop codon allowing for the translation of PIII fusion proteins. The sequence numbering corresponds to the maturation library, matLib. (b) Schematic rendering of the Alphabody scaffold as a single-chain three-helix antiparallel coiled-coil. (c) Schematic representation of the interactions between the core residues in an antiparallel coiled-coil. Hydrophobic interactions between isoleucines at positions a and d in each heptad repeat stabilize the Alphabody fold. Additional stabilizing interactions can arise from amino acids at positions e and g in helices A and C, positions g and g in helices B and C and positions e and e in helices A and B.