Decoupling of Interactions between Model-Charged Peptides Reveals Key Factors Responsible for Liquid-Liquid Phase Separation.

Liquid-liquid phase separation (LLPS) by disordered proteins has been shown to govern biological processes and cause numerous diseases. Therefore, a deeper understanding of the interactions and their variation with external factors is key to modulating the LLPS behavior of different systems and protecting proteins from pathological aggregation. In this context, we have looked at interactions between similarly charged peptides to understand the molecular features that may drive or prevent condensate formation under various conditions. We have studied dimer formation for model peptides where charged and noncharged amino acids have been placed alternatively. Using arginine and glutamic acid as the charged residues and varying the other residues with glycine, alanine, and proline to alter hydrophobicity, we have obtained the free-energy surface (FES) for the dimer formation for these systems under high salt concentration at two different temperatures using all-atom molecular dynamics simulations and the well-tempered metadynamics method. Our results indicate that a combination of effects such as hydrophobicity, arginine-arginine interactions, or water release from the solvation shell makes dimerization free energy more favorable for the positively charged peptides with lower flexibility. For the negatively charged peptides, the crucial role of water has been found in governing the FES. Systems having charged residues and phenylalanine in the peptide sequence also have been studied at high salt concentrations using unbiased simulations. In this case, only the positively charged peptides were found to aggregate through temperature-dependent hydrophobic and cation-π interactions. Overall, our study indicates that the negatively charged peptides are more likely to remain in the dilute phase under various conditions compared to the positively charged systems. The findings from our study would be helpful in designing and controlling systems to obtain LLPS behavior for therapeutic usage.