Pressure and Temperature Phase Diagram for Liquid–Liquid Phase Separation of the RNA-Binding Protein Fused in Sarcoma

• Published in: The journal of physical chemistry. B Vol. 125; no. 25; pp. 6821 - 6829
• Main Authors: Li, Shujie, Yoshizawa, Takuya, Yamazaki, Ryota, Fujiwara, Ayano, Kameda, Tomoshi, Kitahara, Ryo
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.07.2021
• Subjects: Amyotrophic Lateral Sclerosis; B: Biophysical and Biochemical Systems and Processes; Humans; Protein Domains; RNA-Binding Protein FUS - chemistry; Temperature
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/acs.jpcb.1c01451

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.