Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation...

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Membraneless organelles form via phase separation of intrinsically disordered proteins (IDPs).
• Understanding sequence determinants of IDP phase separation is vital for cellular function, disease, and bioinspired materials.
• The RGG domain of LAF-1 is a model system for studying protein phase separation.

Purpose of the Study:

• To identify and characterize sequence features governing IDP phase separation using the LAF-1 RGG domain.
• To investigate the role of charge patterning and specific residues (tyrosine, arginine) in phase behavior.
• To explore the impact of sequence modifications on the biophysical properties of condensates.

Main Methods:

• Coarse-grained modeling to predict protein regions involved in phase separation.
• Site-directed mutagenesis and sequence shuffling to alter charge patterning.
• In vitro and in vivo assays to assess phase separation.
• All-atom simulations to analyze residue contributions and interaction modes.

Main Results:

• A highly conserved, high-contact probability region within the RGG domain was identified; its deletion disrupted phase separation.
• Sequences with segregated charges exhibited increased phase separation propensity, suggesting natural sequences are selected to moderate this.
• Tyrosine and arginine residues were confirmed to be important for phase separation.
• Perturbed RGG-derived condensates maintained liquid-like properties.

Conclusions:

• Specific sequence features, including conserved regions and charge patterning, critically regulate IDP phase separation.
• The findings provide insights into the biophysical principles governing biomolecular condensation.
• This work advances the understanding of IDP behavior in cellular organization and disease.