Intra-Protein Interfaces Control Folding Dynamics and Mechanical Stability in a De Novo Designed Repeat Protein

Protein design has made it possible to generate stable structures with high accuracy, but the principles that determine how designed proteins fold and respond to force remain less understood. Repeat proteins provide a powerful test case because their stability is distributed across modular structural units and nearest-neighbor interfaces rather than concentrated in a single hydrophobic core. Here, we used single-molecule optical tweezers, intramolecular crosslinking and molecular dynamics simulations to dissect the folding landscape of DHR14R, a de novo designed helical repeat protein with a deep-learning-redesigned sequence. By introducing site-specific intramolecular crosslinks, we imposed defined boundary conditions on the repeat array and directly manipulated its mechanical folding pathway. DHR14R unfolds reversibly from its mechanically weakest boundary, the N-terminus, through a series of discrete intermediates. Refolding begins with the formation of a three-helix seed rather than a complete two-helix repeat, showing that the structural repeat is not the elementary cooperative folding unit. Preformed terminal interfaces accelerate folding, indicating that seed formation is rate-limiting. Stabilizing both termini eliminates the low-force terminal-fraying pathway and forces disruption of internal interfaces, increasing mechanical stability more than twofold. Together, these results show that designed repeat-protein folding is governed by seed formation, interface propagation, and terminal boundary conditions. They establish intramolecular crosslinking as a strategy for rationally reshaping folding landscapes in designed proteins.