Frustration Drives the Folding Dynamics of Repeat Proteins

Ankyrin repeat (AR) proteins consist of tandem ~33-residue motifs and frequently mediate protein–protein interactions in diverse biological systems. These proteins typically adopt elongated, non-globular shapes, and their folding is governed by shortrange contacts, with each repeat stabilized by neighboring interactions. Here, we investigate the folding landscape of the AR domain of IκBα, a cooperative unit of six repeats (AR1–AR6), where AR5 and AR6 exhibit reduced stability. Despite its simple architecture, IκBα follows a complex folding trajectory through sequential on-pathway high-energy intermediates. Mutations aligning with AR consensus motifs generally enhance folding stability, whereas deviations compromise it. To dissect these effects, we combine unbiased and biased coarse-grained AWSEM simulations with the Energy Landscape Visualization Method (ELViM), which projects the globally funneled energy surface of three sequences: WT, a fast-folding mutant (V93L), and a slow-folding mutant (L131V), into a low-dimensional representation. This analysis reveals preferred folding paths for each variant, highlights the role of energetic frustration, and shows that consensus-aligned mutations reduce frustration and promote proper folding. Finally, we demonstrate that reversing destabilizing mutations helps avoid kinetic traps. Our study highlights the utility of ELViM for visualizing complex energy landscapes and informing strategies to enhance folding robustness in repeat proteins