Macromolecular Crowding Promotes Reentrant Liquid-Liquid Phase Separation of Human Serum Transferrin and Prevents Surface-Induced Fibrillation

Abstract

AbstractProtein aggregation and inactivation upon surface immobilization are major limiting factors for analytical applications in biotechnology related fields. Protein immobilization on solid surfaces often requires multi-step 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 3–20) 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 (0–3 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.