In Silico Fold-Switching Protein Design Driven by Cα-Based Statistical Potential

Structural plasticity of naturally occurring proteins allows them to change their shape in response to environmental factors such as pH, temperature, or binding partners. This ability to adopt different conformations is essential for many biological processes. While computational methods have been applied to design and redesign protein sequences that fold to a single ordered and stable state, the computational design of protein sequences with high sequence similarity that adopt well-defined but structurally divergent structures remains an outstanding challenge. Here, we designed 28 pairs of sequences using Monte Carlo simulation, denoted as (a₁, b₁), (a₂, b₂), (a₃, b₃), ..., (a₂₈, b₂₈), where a_i and b_i represent sequences adopting the 3-α fold and 4β + α fold, respectively. Among these, we identified three sets of fold-switching protein sequences, (a₁, b₁), (a₂, b₂), and (a₃, b₃): one with 89.29% sequence similarity and two others with 87.50% sequence similarity. This reflects the ability of statistical potential to finely balance competing structural constraints. The designed sequences differ by only few residues; however, they possess different tertiary structures: a 3-α helix fold and a 4β + α fold. In addition, sequence variants for a₁, a₂, and a₃ are also designed using rational design guided by sequence analysis, and the results show striking outcomes: single point mutations, specifically D26C or A39F in a₁, are sufficient to induce fold switching from the 3-α fold to the 4β + α fold while maintaining 98% sequence similarity with the parent sequence. Together, these findings suggest that the design approach is successful in designing fold-switching sequences that are compatible with their respective target structures. This work also ensures that the developed one-body and two-body statistical potentials are successful in designing protein sequences that exhibit fold conservation and the fold-switching phenomenon, as well as stability at the respective target structures.