Hydrophobicity of arginine leads to reentrant liquid-liquid phase separation behaviors of arginine-rich proteins

Abstract

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.

Liquid–Liquid Phase Separation Modifies the Dynamic Properties of Intrinsically Disordered Proteins

“…EPR was also used to study IDP dynamics and in arginine-rich peptides. 39 Along with experimental methods, computational approaches have been used to investigate protein condensates. 11,40−44 While coarse-grained methods 45−47 have been successfully exploited to reproduce condensate properties, atomistic simulation 48 can also provide detailed biophysical insight into the equilibrated condensed phase at atomic resolution.…”

“…Reorientational dynamics of IDPs under phase-separating conditions have been investigated using time-resolved fluorescence spectroscopy techniques, for example, in α-synuclein, where decreased chain flexibility was observed in LLPS compared to dilute conditions, and tau protein, where fluorescence and EPR spectroscopies , were used to characterize modulation of rotational correlation times upon phase separation. EPR was also used to study IDP dynamics and in arginine-rich peptides . Along with experimental methods, computational approaches have been used to investigate protein condensates. ,− While coarse-grained methods − have been successfully exploited to reproduce condensate properties, atomistic simulation can also provide detailed biophysical insight into the equilibrated condensed phase at atomic resolution.…”

Phase Separation and Ageing of Glycine-Rich Protein from Tick Adhesive

“…In addition to that, the five aromatic residues are fairly well-spaced and such equally spaced aromatic residues have been proposed to form liquid condensates 50 . Also, arginine (prominent in the C-terminus) has been proven to be relatively more hydrophobic than lysine (prominent in the N-terminus) due to the presence of π-electron-rich guanidium group which allows them to form π-π bonds along with cation-π bonds 51 . Finally, simple coacervation is more favorable if the protein contains relatively hydrophobic stickers and polar spacers 52 , which indeed seems to be the case for the C-terminus with multiple serine, asparagine, and glutamine residues.…”

“…In addition to that, the five aromatic residues are fairly well-spaced and such equally spaced aromatic residues have been proposed to form liquid condensates 50 . Also, arginine (prominent in the C-terminus) has been proven to be relatively more hydrophobic than lysine (prominent in the N-terminus) due to the presence of πelectron-rich guanidium group which allows them to form π-π bonds along with cation-π bonds 51 .…”

Polyelectrolyte-multivalent molecule complexes: physicochemical properties and applications

“…43,[242][243][244] In addition, the intracellular molecular crowding can be imitated and tuned by adding water-attracting and dehydrating additives (such as salt and polyethylene glycol) that allow increasing the effective concentration of the macromolecules in complex coacervates. 245,246 In parallel, advances in experimental techniques -in particular advanced microscopies -are making it possible to overcome the limitations when it comes to studying and quantifying complex coacervates both in vitro and in vivo conditions. In this framework, it is worth to highlight the tremendous advances in quantitative phase imaging (QPI), a powerful label-free wide-field microscopy approach to characterize and quantify (in real time) biomacromolecules in liquid condensates.…”