Mechanism of 1,6-hexanediol-induced protein droplet dissolution: Thermodynamic insights from amino acid solubility

Protein solutions can form droplets through liquid-liquid phase separation, a phenomenon involved in various intracellular processes. The aliphatic alcohol, 1,6-hexanediol (1,6-HD), known for its ability to dissolve protein droplets, is commonly used to distinguish proteinaceous liquid droplets from aggregates. However, the thermodynamic basis of 1,6-HD-induced droplet dissolution, particularly at the amino acid level, remains unclear. This study investigated the underlying thermodynamic mechanism, focusing on interactions between proteinogenic amino acid residues and 1,6-HD. Temperature-dependent solubility measurements of amino acid derivatives (N-acetyl-l-amino acid amides) revealed that both aromatic and aliphatic species were more soluble in 20 wt% aqueous 1,6-HD solution than in water, indicating their thermodynamic stabilization; particularly, N-acetyl-l-tryptophan amide was strongly stabilized. An entropy-enthalpy compensation plot demonstrated an entropy-driven stabilization of hydrophobic amino acid side chains. NMR spectra suggested that this stabilization was attributed to enhanced side-chain mobility upon 1,6-HD addition. Consistent with these findings, 1,6-HD also induced the droplet dissolution of N-acetyl-l-amino acid ethyl esters. These results indicate that protein droplet dissolution by 1,6-HD is driven by the entropy-driven stabilization of hydrophobic amino acid residues.