Determinants of Protein Folding Pathways: Lessons from Metamorphic Proteins

The protein folding problem has traditionally been defined by two complementary challenges: predicting the three-dimensional structure of a protein from its amino acid sequence and understanding the mechanism by which this structure is attained. While recent advances in artificial intelligence have largely addressed the former, the latter remains unresolved. Early studies showed that many small proteins fold in a cooperative two-state manner, shifting attention toward transition states and energy landscapes. Comparative analyses of protein families further revealed that folding mechanisms are often conserved among proteins sharing the same topology, suggesting a dominant role of structure in shaping folding pathways. However, this framework does not explain when and how a protein commits to a specific topology. Metamorphic proteins, in which highly similar sequences adopt distinct native folds, provide a powerful complementary approach. Studies of these systems show that closely related sequences can follow different folding mechanisms without sharing common intermediates. These findings indicate that folding pathways are determined at very early stages and are encoded within the denatured ensemble through subtle structural and energetic biases. Here, we review the evolution of protein folding studies and propose a unified view in which folding mechanisms are selected early, with the denatured state playing a central role in defining both folding pathways and final topology.