Comparative roles of charge, π , and hydrophobic interactions in sequence-dependent phase separation of intrinsically disordered proteins

Abstract

Endeavoring toward a transferable, predictive coarse-grained explicit-chain model for biomolecular condensates underlain by liquid–liquid phase separation (LLPS) of proteins, we conducted multiple-chain simulations of the N-terminal intrinsically disordered region (IDR) of DEAD-box helicase Ddx4, as a test case, to assess roles of electrostatic, hydrophobic, cation–π, and aromatic interactions in amino acid sequence-dependent LLPS. We evaluated three different residue–residue interaction schemes with a shared electrostatic potential. Neither a common hydrophobicity scheme nor one augmented with arginine/lysine-aromatic cation–π interactions consistently accounted for available experimental LLPS data on the wild-type, a charge-scrambled, a phenylalanine-to-alanine (FtoA), and an arginine-to-lysine (RtoK) mutant of Ddx4 IDR. In contrast, interactions based on contact statistics among folded globular protein structures reproduce the overall experimental trend, including that the RtoK mutant has a much diminished LLPS propensity. Consistency between simulation and experiment was also found for RtoK mutants of P-granule protein LAF-1, underscoring that, to a degree, important LLPS-driving π-related interactions are embodied in classical statistical potentials. Further elucidation is necessary, however, especially of phenylalanine’s role in condensate assembly because experiments on FtoA and tyrosine-to-phenylalanine mutants suggest that LLPS-driving phenylalanine interactions are significantly weaker than posited by common statistical potentials. Protein–protein electrostatic interactions are modulated by relative permittivity, which in general depends on aqueous protein concentration. Analytical theory suggests that this dependence entails enhanced interprotein interactions in the condensed phase but more favorable protein–solvent interactions in the dilute phase. The opposing trends lead to only a modest overall impact on LLPS.

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

“…These findings suggested that enhanced cation–π and electrostatic interactions were required to achieve a qualitative agreement with the FUS experimental behavior. When we included an additional term in the potential energy to increase the strength of cation–π interactions at low salt, as recently proposed 121 , we qualitatively recover a higher critical temperature for full FUS versus its PLD. Our PMFs indicate that when transitioning from low to moderate salt, electrostatic interactions diminish significantly, while hydrophobic interactions remain strong, giving rise to reentrant phase behavior.…”

RNA modulation of transport properties and stability in phase separated condensates

“…Here, we investigate the dual effect of poly-U RNA in controlling the stability and dynamics of RNAbinding protein condensates. By means of Molecular Dynamics simulations of a novel sequence dependent coarse-grained model for proteins and RNA 97,111,119 , we FIG. 5.…”

Biophysics of Phase Separation of Disordered Proteins Is Governed by Balance between Short- And Long-Range Interactions

“… 20 − 22 Major molecular contributions that govern the condensate properties have been assigned to charge–charge, cation−π, and π–π interactions. 23 − 26 These studies showed that the composition as well as the organization 14 , 27 − 30 of charged and aromatic/hydrophobic residues can strongly affect LLPS. These molecular interactions entropically stabilize the condensate, and the balance between their enthalpy and entropy components 31 , 32 dictates the nature of the condensates.…”