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Figure 1
Design of the helix bundle–DARPin–apoferritin–helix bundle scaffold. (a) The scaffold is depicted as a cartoon, with apoferritin forming the inner cage and the DARPin serving as a platform for binding small cargo proteins on the outer portion. (b) The screening process for DARPin–apoferritin fusion constructs (model 1–6) is illustrated, with each construct and its junction sequences presented in distinct colors. (c, d) A detailed view of (c) model 4 and (d) model 6, specifically designed and characterized in this study, are shown. The apoferritin core of the scaffold, colored in gray, is prominently displayed as 24 subunits arranged in octahedral 432 symmetry. The N-terminal α-helix of apoferritin is connected to the C-terminal α-helix of the DARPin subunit (cyan in model 4 and magenta in model 6) through a continuous α-helix depicted in blue. The variable cargo-binding surface of the DARPin is highlighted in orange. In model 4, four neighboring DARPin molecules are oriented towards the C4 channel of the apoferritin cage. The helix-bundle part is designed to stabilize the scaffold at the N-terminus of the DARPin (deep salmon) and the C-terminus of apoferritin (yellow), as shown in (c) (right). Additionally, several mutations are introduced at the interface between the DARPin and apoferritin to enhance rigidity. In model 6, the N-termini of two DARPin subunits tightly bind around the C3 channel of apoferritin due to the extended and rotated α-helical fusion compared with that in model 4 (d).

IUCrJ
Volume 12| Part 3| May 2025| Pages 393-402
ISSN: 2052-2525
https://doi.org/10.1107/S2052252525003021