Residue-level mapping of crowding effects on protein phase separation

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Protein liquid-liquid phase separation (LLPS) is crucial for cellular organization.
• The impact of the cellular crowded environment on protein LLPS is not fully understood.
• How cellular crowding shapes the chemical environment and affects protein LLPS remains largely unknown.

Purpose of the Study:

• To investigate residue-level crowding effects on the intrinsically disordered region of RNA polymerase II.
• To understand how different types of crowders (polymer-based, protein-based, and reconstituted cytosol) influence the chemical environment and protein interactions.
• To develop a framework for probing residue-level influences in cell-like environments.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy was employed to map residue-level crowding effects.
• Experiments were conducted using polymer-based crowders, protein-based crowders, and reconstituted E. coli cytosol.
• Analysis focused on changes in chemical environment and protein self-interactions.

Main Results:

• A general trend of enhanced protein self-interactions was observed across all tested crowding conditions.
• Distinct chemical environments were identified, which varied depending on the crowder's identity.
• These variations reflect changes in preferential interactions between proteins and crowders.

Conclusions:

• Cellular crowding significantly impacts protein phase separation by altering the chemical environment and protein interactions.
• The identity of crowders plays a critical role in shaping these effects.
• The findings provide a strategy for evaluating the chemical influence of crowders and designing more physiologically relevant in vitro crowding models.