Comprehensive Biophysical Profiling Evidences Self-Oligomerization of Bacterially Expressed Pc Protein.

Area of Science:

• Epigenetics
• Molecular Biology
• Structural Biology

Background:

• Polycomb (Pc) protein is essential for Polycomb Repressive Complex 1 (PRC1) mediated epigenetic gene silencing.
• Structural characterization of Pc is challenging due to its intrinsic disorder and self-association properties.

Purpose of the Study:

• To produce and biophysically characterize Drosophila melanogaster Pc constructs for structural studies.
• To investigate the role of intrinsic disorder, oligomerization, and self-association in Pc function.

Main Methods:

• Recombinant protein production and purification using solubility-enhancing fusion strategies.
• Biophysical characterization including circular dichroism, thermal shift assays, nanoDSF, DLS, and SEC-MALS.
• Cryo-electron microscopy and AlphaFold-based modeling.

Main Results:

• Milligram quantities of stable, MBP-tagged Pc protein were obtained.
• Pc is predominantly intrinsically disordered outside its chromodomain, with conditional folding upon oligomerization.
• Pc exhibits buffer-dependent stability and concentration-dependent self-association into heterogeneous assemblies.
• Cryo-EM and modeling suggest a conditional folding mechanism linked to self-association.

Conclusions:

• A robust framework for Pc biophysical investigation was established.
• Intrinsic disorder, oligomerization, and self-association are key mechanistic features of Polycomb-mediated chromatin regulation.