Tunable multiphase dynamics of arginine and lysine liquid condensates

Abstract

Liquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease. The design principles underlying biomolecular phase separation have the potential to drive the development of novel liquid-based organelles and therapeutics, however, an understanding of how individual molecules contribute to emergent material properties, and approaches to directly manipulate phase dynamics are lacking. Here, using microrheology, we demonstrate that droplets of poly-arginine coassembled with mono/polynucleotides have approximately 100 fold greater viscosity than comparable lysine droplets, both of which can be finer tuned by polymer length. We find that these amino acid-level differences can drive the formation of coexisting immiscible phases with tunable formation kinetics and can be further exploited to trigger the controlled release of droplet components. Together, this work provides a novel mechanism for leveraging sequence-level components in order to regulate droplet dynamics and multiphase coexistence.

Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions

“…Glu, Asp, and nucleotides also have charged groups and π-orbitals, and hence, their homotypic interaction could similarly exhibit a transition from repulsion to the attraction as salt increases. Moreover, π–π bonding between Arg and nucleotides is consistent with the much higher salt-range stability observed experimentally for droplets co-assembled with poly-Arg and polynucleotides over those formed with poly-Lys and polynucleotides 89 . These unexpected results further illuminate the differences in the molecular interactions stabilizing protein condensates at high salt versus low salt and put forward Arg–Arg as an additional force that stabilizes the homotypic LLPS of PR 25 in the high-salt regime.…”

“…2 ). Both the significant strengthening with salt and the high magnitude of the Arg–Tyr/Phe interaction throughout are explained by a strong contribution of π–π bonding between the π–orbitals in the sp 2 -hybridized guanidinium group of Arg and the π–electrons of the aromatic rings 86 – 89 . This important π–π contribution was further demonstrated by performing additional PMF calculations in which the sidechain π–orbitals of Arg and Tyr were maintained in either a parallel (Supplementary Fig.…”

Valency and Binding Affinity Variations Can Regulate the Multilayered Organization of Protein Condensates with Many Components

“…Simultaneously, positioning the low-valency species towards the surface decreases the energetic penalty for interface formation (interfacial free energy minimization). The key role of the surface tension in driving multilayered condensate organization has been reported for in vitro complex coacervates [ 47 , 50 ]. Moreover, our findings provide a thermodynamic explanation for the scaffold-client model [ 40 ], which proposes that nucleation of biomolecular condensates with many components is driven by interactions between high-valency proteins.…”

“…Experiments and simulations propose that the multilayered organization of the nucleolus emerges from differences in the surface tensions of the various phases [ 15 ]. The importance of surface tension in driving a multilayered molecular organization of condensates has been corroborated for in vitro complex coacervates that form condensates with up to three layers [ 47 , 50 ]. Competing interactions among protein–RNA networks also play a role in driving the formation of multiphase condensates [ 33 ].…”

Deciphering how naturally occurring sequence features impact the phase behaviors of disordered prion-like domains

“…1f). These analyses were motivated by results that highlight the role of Arg as a sticker 16 , the relative weakness of Lys as a sticker 16, 22, 23, 31, 32 , the contributions of charged residues that destabilize phase separation either as spacers that promote solvation 27, 28 or via electrostatic repulsions 33 , and the suggestion that Tyr is likely to be a stronger sticker than Phe 16, 34 .…”