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

Abstract

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 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 (residues 3–20) containing hydrophobic amino acids. The present LLPS of Tf is a rare example of salt-mediated re-entrant 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 compact aqueous compartments of biomolecular condensates with a substantial amount of interfacial water preserve the structure and functionality of Tf. 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.

Biomolecular Condensation of Trypsin Prevents Autolysis and Promotes Ca2+-Mediated Activation of Esterase Activity

“…These assemblies in the presence of PEG display fusion, surface wetting, and dripping phenomena authenticating their liquid-like droplet nature (Figure 1B). [45][46][47] Similar liquid-like droplet/condensate formation has been reported previously for a wide range of proteins in the absence or presence of crowders via spontaneous LLPS. [45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64] Notably, trypsin from porcine and α-chymotrypsin also exhibit LLPS in the presence of 10% PEG to yield liquid-like condensates (Figure S2).…”

“…Recent studies have illustrated that inert synthetic as well as protein crowders can promote LLPS of ordered proteins via promoting multivalent soft protein-protein interactions. [45][46][47][48][49] Notably, the polypeptide chain of trypsin contains neither any low complexity domains (LCDs) nor any disordered regions as revealed from Simple Modular Architecture Research Tool (SMART) and IUPred2 sequence prediction algorithms, respectively (Scheme 1C). 66 Initially, we seek to know whether macromolecular crowding promotes intermolecular protein-protein interactions of trypsin under physiological conditions using confocal laser scanning microscopy (CLSM).…”

“…Lately, it has also been observed that macromolecular crowding promotes liquid-liquid phase separation (LLPS) of many ordered proteins and functional enzymes. [45][46][47][48][49] In general, proteins containing intrinsically disordered regions (IDRs) with low complexity domains (LCDs) have been shown to undergo LLPS in the absence and presence of inert crowders. [50][51][52][53][54][55][56][57][58][59][60][61][62][63][64] LLPS is a spontaneous and thermodynamically favorable liquid-liquid demixing phenomenon for proteins which exhibit weak multivalent attractive intermolecular protein-protein interactions.…”

“…Macromolecular crowding promotes these intermolecular interactions via excluded volume effect by lowering the critical concentration of proteins required for phase separation. Due to their highly dynamic and liquidlike nature, biomolecular condensates are highly sensitive towards several factors including animo acid composition, concentration, ionic strength, pH, temperature, pressure, and [45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64] In the present study, we have investigated the impact of macromolecular crowding on the activity of trypsin in the absence and presence of Ca 2+ to understand the regulatory role of Ca 2+ in the esterase activity of trypsin-catalyzed hydrolysis of p-nitrophenyl acetate (p-NPA) (Scheme 1B). The p-NPA hydrolysis by trypsin is a model enzymatic reaction which can be conveniently followed by UV-visible spectroscopy.…”

Imaging-Based Quantitative Assessment of Biomolecular Condensatesin vitroand in Cells

“…Whilst in this study we used PEG, 1,6-HD, and salts to modulate the hydrophobic interactions that are important to the formation of Nup100FG particles, similar studies could be performed for other proteins that rely on other interaction types, such as electrostatic, cation-pi or polar interactions. These kinds of studies could aid in better linking the observed changes to the molecular driving forces controlling the formation and material properties of the particles (79,87). Moreover, thorough mapping of how particles respond to external stimuli can also aid in improving our understanding of the adaptability and stability of condensates in the cellular context.…”