Macromolecular crowding has a profound impact on the conformational dynamics and intermolecular interactions of biological macromolecules. In this context, the role of inert synthetic crowders in the protein−protein interactions of globular proteins is poorly understood. Here, using native human serum albumin (HSA) under physiological conditions, we show that macromolecular crowding induces liquid−liquid phase separation (LLPS) via liquid-like membrane-less droplet formation in a concentration-and time-dependent manner. Circular dichroism measurements reveal significant alteration in the secondary structure of HSA inside the droplet during aging. In contrast, at a high protein concentration, a liquid-to-solid-like phase transition has been observed upon maturation. Our findings reveal that the LLPS of HSA is mainly driven by enthalpically controlled intermolecular 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 of soft protein−protein interactions of globular proteins in a crowded cellular environment in driving the LLPS.

Protein aggregation and inactivation upon surface immobilization are major limiting factors for analytical applications in biotechnology related fields. Protein immobilization on solid surfaces often requires multistep surface passivation which is time consuming and inefficient. Herein, we have discovered that biomolecular condensates of biologically active human serum transferrin (Tf) can effectively prevent surface induced fibrillation and preserve the native like conformation of phase separated Tf over a period of 30 days. It has been observed that macromolecular crowding promotes homotypic liquid liquid phase separation (LLPS) of Tf through enthalpically driven multivalent hydrophobic interactions possibly via the involvement of its low complexity domain (residue 3 to 20) containing hydrophobic amino acids. The present LLPS of Tf is a rare example of salt-mediated reentrant phase separation in a broad range of salt concentrations (0 to 3 M) solely via the involvement of hydrophobic interactions. Notably, no liquid to solid like phase transition has been observed over a period of 30 days, suggesting the intact conformational integrity of phase separated Tf as revealed from single droplet Raman, circular dichroism, and Fourier transform infrared spectroscopy measurements. More importantly, we discovered that the phase separated condensates of Tf completely inhibit the surface induced fibrillation of Tf, illustrating the protective role of these liquid-like condensates against denaturation and aggregation of biomolecules. The cell mimicking aqueous compartments of biomolecular condensates with a substantial amount of interfacial water preserve the structure and functionality of biomolecules. Our present study highlights an important functional aspect of biologically active protein condensates and may have wide ranging implications in cell physiology and biotechnological applications.