Unraveling the complexity of Exendin-4 folding through two distinct pathways

Protein folding is a prominent area of research in the life sciences. Exendin-4, a 39-amino acid peptide hormone, is of particular interest due to its potential therapeutic applications. In this study, we employed a steered molecular dynamics method to investigate the folding of Exendin-4. Our simulations reveal an intermediate state during the folding process, suggesting a more complex three-state mode of folding. Structural analysis indicates that Exendin-4 folds through two distinct pathways: pathway 1 involves gradual growth of a helical structure after the collapse of the hydrophobic core in the Trp-cage, while pathway 2 involves initial formation of local microdomains (helical structure and Trp-cage) which then combine with each other to form a stable native structure. We found that the folding along these pathways follows the hydrophobic collapse mechanism and the diffusion-collision mechanism, respectively. We believe that these two mechanisms together govern the folding of Exendin-4. These results significantly differ from those observed in most small protein folding. Our study on the folding of Exendin-4 will advance our understanding of the protein folding mechanism.