Local Frustration Modulates the Folding Dynamics of a Repeat Protein

Abstract

Repeat proteins fold through pathways that are strongly shaped by local energetics, making them highly sensitive to mutations. The ankyrin repeat (AR) domain of IκBα is a cooperative folding unit in which the first four repeats (AR1–AR4) are stable, while the last two are destabilized. Despite this simple modular architecture, IκBα follows a complex folding trajectory involving high-energy intermediates. Here, we investigate how sequence variations modulate folding pathways using coarse-grained AWSEM simulations combined with the Energy Landscape Visualization Method (ELViM) and local frustration analysis. We compare the wild type (WT) with two consensus-designed variants: V93L, which accelerates folding, and L131V, which destabilizes and slows down folding of the protein in vitro. Our results show that WT and V93L share a similar folding funnel, though V93L folds more directly into the native state. In contrast, L131V reshapes the landscape by stabilizing non-native kinetic trap with minimally frustrated contacts. Reversing the L131V mutation allows the protein to reach the native conformation, whereas maintaining it confines the protein to misfolded states that can only be escaped at very high temperatures, without productive folding. These findings highlight how subtle sequence changes can tune frustration, modulate kinetic trapping, and control folding efficiency in repeat proteins.