Macromolecular Crowding-Induced Unusual Liquid-Liquid PhaseSeparation of Human Serum Albumin via Soft Protein-ProteinInteractions

Macromolecular crowding has a profound impacton the conformational dynamics and intermolecular interactions ofbiological macromolecules. In this context, the role of inertsynthetic crowders in the protein-protein interactions of globularproteins is poorly understood. Here, using native human serumalbumin (HSA) under physiological conditions, we show thatmacromolecular crowding induces liquid-liquid phase separation(LLPS) via liquid-like membrane-less droplet formation in aconcentration- and time-dependent manner. Circular dichroismmeasurements reveal significant alteration in the secondarystructure of HSA inside the droplet during aging. In contrast, ata high protein concentration, a liquid-to-solid-like phase transitionhas been observed upon maturation. Ourfindings reveal that the LLPS of HSA is mainly driven by enthalpically controlledintermolecular protein-protein interactions via hydrophobic contacts involving aromatic and/or nonaromatic residues. Moreover,modulation of LLPS of HSA has been demonstrated upon denaturation and ligand binding. This study highlights the importance ofsoft protein-protein interactions of globular proteins in a crowded cellular environment in driving the LLPS.

Automated summary

Macromolecular crowding induces liquid-liquid phase separation in biological macromolecules, leading to the formation of liquid-like membrane-less droplets. This study reveals significant alteration in the secondary structure of the macromolecules inside the droplets. At high protein concentrations, a liquid-to-solid-like phase transition is observed. The study highlights the importance of enthalpically controlled protein-protein interactions in driving the phase separation.