Liquid–liquid phase separation (LLPS) of proteins and nucleic acids to form membraneless cellular compartments is considered to be involved in various biological functions. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vivo and in vitro. Here, we investigated the effects of pressure and temperature on the LLPS of FUS by high-pressure microscopy and high-pressure UV/vis spectroscopy. The phase-separated condensate of FUS was obliterated with increasing pressure but was observed again at a higher pressure. We generated a pressure–temperature phase diagram that describes the phase separation of FUS and provides a general understanding of the thermodynamic properties of self-assembly and phase separation of proteins. FUS has two types of condensed phases, observed at low pressure (LP-LLPS) and high pressure (HP-LLPS). The HP-LLPS state was more condensed and exhibited lower susceptibility to dissolution by 1,6-hexanediol and karyopherin-β2 than the LP-LLPS state. Moreover, molecular dynamic simulations revealed that electrostatic interactions were destabilized, whereas cation−π, π–π, and hydrophobic interactions were stabilized in HP-LLPS. When cation−π, π–π, and hydrophobic interactions were transiently stabilized in the cellular environment, the phase transition to HP-LLPS occurred; this might be correlated to the aberrant enrichment of cytoplasmic ribonucleoprotein granules, leading to amyotrophic lateral sclerosis.

Host–guest interactions between naphthalene-based molecular tubes and small molecules have been studied to understand selective recognition. However, the volumetric properties of complexation remain largely unknown. In this study, we investigated the volumetric properties for the binding of 1,4-dioxane to a pair of naphthotubes (i.e., anti- and syn-isomers), each possessing two inwardly directed amide groups in the hydrophobic cavity, using nuclear magnetic resonance and fluorescence spectroscopy coupled with pressure perturbation. We found that the partial molar volume change for the association of 1,4-dioxane with the naphthotube was −6.3 ± 0.1 mL/mol for the anti-isomer and 3.2 ± 0.4 mL/mol for the syn-isomer. Moreover, the hydrogen bonds of the naphthotubes with 1,4-dioxane were less compressible than those with water molecules, indicating that more rigid hydrogen bonds existed in the complexes with 1,4-dioxane. Molecular dynamics simulations showed that one opening of the cavity in the syn-isomer was widened because of the repulsion between the four COO– charges, which allowed more water molecules to access the hydrophobic cavity than in the case of the anti-isomer. The difference in the partial molar volume change was explained by variations in the hydration of naphthotube hydrophobic cavities. The enhanced understanding of the molecular basis of volume changes during 1,4-dioxane–naphthotube complexation may provide insights into ligand binding to bioreceptors.