N-acetyl amino acid amide solubility in aqueous 1,6-hexanediol solutions: Insights into the protein droplet deformation mechanism.

Proteinaceous liquid droplets, generated by liquid-liquid phase separation, function as membraneless compartments that are essential for diverse biological functions. Studies addressing droplet generation have used 1,6-hexanediol (1,6-HD) as a droplet-discerning agent owing to its capacity to induce droplet deformation. Despite the empirical utility of 1,6-HD, the mechanism underlying 1,6-HD-induced droplet deformation remains unknown. In this study, the solubilities of N-acetyl amino acid amides, which correspond to proteinogenic amino acid residues, were examined in the presence of 1,6-HD at 25 °C. Other solvents included ethanol, 1-propanol, and amides. Remarkably, 1,6-HD effectively solubilized hydrophobic species (particularly aromatic species) and exhibited reduced efficacy in solubilizing hydrophilic species and peptide bond moieties. These solubilizing effects are reflected in changes in protein solubility and structure. Specifically, 1,6-HD primarily targets the hydrophobic regions of a protein, increasing protein solubility without causing substantial structural changes. This solubilization mechanism is essential for elucidating the role of 1,6-HD as a droplet-discerning agent and recognizing its potential limitations.