Phase separation of a bacterial disordered chaperone mediated by electrostatic interactions.

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Liquid-liquid phase separation (LLPS) is crucial for cellular organization and stress response.
• LLPS is well-studied in intrinsically disordered proteins, but less is known about highly charged, intrinsically disordered chaperones.
• The role of LLPS in the function of molecular chaperones, particularly under stress conditions, requires further investigation.

Purpose of the Study:

• To investigate whether highly charged, intrinsically disordered molecular chaperones undergo LLPS.
• To characterize the driving forces and regulatory factors of LLPS in the Escherichia coli acid shock protein Asr.
• To explore the in vivo relevance and functional implications of LLPS in molecular chaperones for cellular homeostasis and stress mitigation.

Main Methods:

• In vitro phase separation assays with purified proteins and nucleic acids.
• Biophysical characterization of condensate properties, including dependence on pH, ionic strength, and polyanion length.
• In vivo imaging of fluorescently tagged chaperones in E. coli cells.
• Fluorescence Recovery After Photobleaching (FRAP) to assess condensate dynamics.
• Analysis of chaperone function in mitigating protein aggregation.

Main Results:

• The highly charged, intrinsically disordered chaperone Asr from E. coli undergoes LLPS driven by electrostatic interactions with polyanions (DNA, RNA, heparin, acidic proteins).
• Asr phase separation is sensitive to charge clusters, polyanion length, pH, and ionic strength.
• In vivo, Asr forms dynamic compartments in E. coli cells, particularly under acidic conditions, which are sensitive to salt concentration.
• Charge-enhanced Asr mutants and homologs form condensates in vivo, co-localizing with acidic proteins and reducing stress-induced aggregation of client proteins.

Conclusions:

• Highly charged, intrinsically disordered molecular chaperones, exemplified by Asr, can participate in LLPS.
• Electrostatic interactions are key drivers for Asr-mediated LLPS, influencing condensate formation and dynamics.
• Asr's LLPS capability serves a functional role in mitigating protein aggregation under cellular stress.
• These findings expand the understanding of LLPS in chaperone biology and suggest potential for engineering biomolecular condensates.