Design Principles for β-Solenoid Stability via Covalent and Electrostatic Capping Motifs.

Extended β-solenoid proteins form highly repetitive architectures representing attractive scaffolds for functional biomaterials, yet their termini are intrinsically prone to fraying, and general stabilization strategies remain elusive. Here we identify covalent and electrostatic capping motifs as orthogonal mechanisms that control β-solenoid stability and environmental response. Using fungal and bacterial ice-nucleating proteins as homologous β-solenoid scaffolds, we disentangle how disulfide-mediated and electrostatic capping govern fold integrity under environmental conditions. Disulfide caps in fungi constrain coil unfolding, preserving β-solenoid function under thermal and pH stress but are selectively vulnerable to reductants, which reduce activity by more than 90%. Charged bacterial termini function via electrostatics and are insensitive to reducing agents yet more susceptible to pH and temperature. These results establish terminal capping chemistry as a key handle to tune the stability and environmental robustness of β-solenoid folds, suggesting a promising strategy for the design of repeat-protein scaffolds.