The Effect of Chain Length and Conformation on the Nucleation of Glycine Homopeptides during the Crystallization Process.

To explore the effect of chain length and conformation on the nucleation of peptides, the primary nucleation induction time of glycine homopeptides in pure water at different supersaturation levels under various temperatures has been determined. Nucleation data suggest that longer chains will prolong the induction time, especially for chains longer than three, where nucleation will occur over several days. In contrast, the nucleation rate increased with an increase in the supersaturation for all homopeptides. Induction time and nucleation difficulty increase at lower temperatures. However, for triglycine, the dihydrate form was produced with an unfolded peptide conformation (pPII) at low temperature. The interfacial energy and activation Gibbs energy of this dihydrate form are both lower than those at high temperature, while the induction time is longer, indicating the classical nucleation theory is not suitable to explain the nucleation phenomenon of triglycine dihydrate. Moreover, gelation and liquid-liquid separation of longer chain glycine homopeptides were observed, which was normally classified to nonclassical nucleation theory. This work provides insight into how the nucleation process evolves with increasing chain length and variable conformation, thereby offering a fundamental understanding of the critical peptide chain length for the classical nucleation theory and complex nucleation process for peptides.