Intrinsically disordered proteins rich in cationic amino acid groups can undergo Liquid-Liquid Phase Separation (LLPS) in the presence of charge-balancing anionic counterparts. Arginine and Lysine are the two most prevalent cationic amino acids in proteins that undergo LLPS, with arginine-rich proteins observed to undergo LLPS more readily than lysine-rich proteins, a feature commonly attributed to arginine’s ability to form stronger cation-π interactions with aromatic groups. Here, we show that arginine’s ability to promote LLPS is independent of the presence of aromatic partners, and that arginine-rich peptides, but not lysine-rich peptides, display re-entrant phase behavior at high salt concentrations. We further demonstrate that the hydrophobicity of arginine is the determining factor giving rise to the reentrant phase behavior and tunable viscoelastic properties of the dense LLPS phase. Controlling arginine-induced reentrant LLPS behavior using temperature and salt concentration opens avenues for the bioengineering of stress-triggered biological phenomena and drug delivery systems.
Coacervation is considered as a ubiquitous mechanism for assembling biomolecular materials outside cells and organizing membraneless organelles inside cells. Despite the importance of mapping binodals to understand the driving forces and thermodynamics of coacervate, quantifying protein concentration within a droplet is significantly challenging owing to its dynamic and viscous nature. A direct imaging‐based method is presented to quantify coacervate using real‐time 3D quantitative phase imaging. The proposed method utilizes the measurements of the refractive index tomograms of individual coacervates and retrieves the protein concentration and volume of individual protein droplets exploiting light‐scattering analysis. The retrievals of accurate protein concentrations are demonstrated in droplets, whereas conventional fluorescence‐based techniques present underestimations. With its simple, direct, real‐time, and quantitative analysis capability, the present method can be utilized in various protein analyses and quantifications for the study of coacervation both in vitro and in vivo.
The load-bearing proteins in mussel holdfasts rely on condensed tris-catecholato-Fe 3+ coordination complexes for their toughness and shockabsorbing properties, and this feature has been successfully translated into synthetic materials with short-term high-performance properties. However, oxidation of catecholic DOPA (3,4-dihydroxyphenylalanine) remains a critical impediment to achieving materials with longer-lasting performance. Here, following the natural mussel pathway for protein processing, we explore how DOPA oxidation impacts coacervation of mussel foot protein-1 (mfp-1) and its capacity for phase-specific metal uptake in vitro. Without metal, DOPA oxidation changed the rheological properties (i.e., viscosity, loss, and storage moduli) of mfp-1 coacervate droplets. However, oxidation-dependent changes were recovered with dithiothreitol (DTT), completely restoring the behavior of mfp-1 coacervates prior to oxidation. With metal, mfp-1 coacervates exhibited gel-like behavior with high viscosity and cohesive forces by forming recognizable bis-and tris-catecholato-Fe complexes, linked to increased energy dissipation and toughness of byssus. These results indicate that Fe 3+ -mediated conversion of liquid−liquid phase-separated polymers into metal-coordinated networks is thorough and rapid, and DTT effectively maintains redox integrity. Our study provides much-needed improvements for processing catechol-functionalized polymers into high-performance materials.
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