The Effect of Polymer Length in Phase Separation

Abstract

Understanding the thermodynamics that drives liquid-liquid phase separation (LLPS) is quite important given the many numbers of diverse biomolecular systems undergoing this phenomenon. Regardless of the diversity, the processes underlying the formation of condensates exhibit physical similarities. Many studies have focused on condensates of long polymers, but very few systems of short polymer condensates have been observed and yet studied. Here we study a short polymer system of various lengths of poly-Adenine RNA and peptide formed by the RGRGG sequence repeats to understand the underlying thermodynamics of LLPS. We carried out MD simulations using the recently developed COCOMO coarse-grained (CG) model which revealed the possibility of condensates for lengths as short as 5-10 residues, which was then confirmed by experiment, making this one of the smallest LLPS systems yet observed. Condensation depends on polymer length and concentration, and phase boundaries were identified. A free energy model was also developed. Results show that the length dependent condensation is driven solely by entropy of confinement and identifies a negative free energy (-ΔG) of phase separation, indicating the stability of the condensates. The simplicity of this system will provide the basis for understanding more biologically realistic systems.

Elucidating the reversible and irreversible self-assembly mechanisms of low-complexity aromatic-rich kinked peptides and steric zipper peptides

“…38 Apart from through maturation or aging (the time-dependent coarsening and loss of dynamicity) of a liquidlike droplet by RNA binding proteins such as FUS, pathogenic fibrils can also be formed by proteins such as Aβ directly through irreversible aggregation without LLPS, which is termed liquid-solid phase separation (LSPS). 39 In addition to full-length proteins and their LC domains, short peptides are able to phase separate, forming liquid condensates [40][41][42][43] and reversible or irreversible fibrils. 12,13,15 Recent crystal structure studies of short peptides have provided different structural properties of the two types of fibrils.…”

“…39 Unlike the irreversible fibrils, the structures of reversible fibrils are organized in kinked strands perpendicular to the fibril spine, or extended β-strands with a hydrous sheet interface, or a stacking pattern with Asp residues from individual β-strands aligned along the fibril spine. 7,12,13,16 For example, fibrils of FUS [37][38][39][40][41][42] (PDB ID: 5XSG) comprise kinked strands without extended β sheets (Fig. S1A †), and fibrils of FUS [54][55][56][57][58][59] (PDB ID: 5XRR) consist of pairs of β-sheets with a hydrophilic interface (Fig.…”

“…[37][38][39][40][41][42] and FUS 54-59 presented increasing assembly capability with increasing scaling factor…”

“…[37][38][39][40][41][42] , FUS 54-59 , Aβ 16-21 and Tau 306-311 , by performing AA-MD and CG-MD simulations. We characterized the self-assembly dynamics of the four hexapeptides and predicted their LLPS/ LSPS propensities.…”