Phase-separation propensity of non-ionic amino acids in peptide-based complex coacervation systems

Abstract

Uncovering the sequence-encoded molecular grammar that governs the liquid–liquid phase separation (LLPS) of proteins is a crucial issue to understand dynamic compartmentalization in living cells and the emergence of protocells. Here, we present a model LLPS system that is induced by electrostatic interactions between anionic nucleic acids and cationic oligolysine peptides modified with 12 different non-ionic amino acids, with the aim of creating an index of ‘phase-separation propensity’ that represents the contribution of non-ionic amino acids to LLPS. Based on turbidimetric titrations and microscopic observations, the lower critical peptide concentrations where LLPS occurs (Ccrit) were determined for each peptide. A correlation analysis between these values and known amino-acid indices unexpectedly showed that eight non-ionic amino acids inhibit the generation of LLPS, whereby the extent of inhibition increases with increasing hydrophobicity of the amino acids. However, three aromatic amino acids deviate from this trend, and rather markedly promote LLPS despite their high hydrophobicity. A comparison with double-stranded DNA and polyacrylic acid revealed that this is primarily due to interactions with DNA nucleobases. Our approach to quantify the contribution of nonionic amino acids can be expected to help to provide a more accurate description and prediction of the LLPS propensity of peptides/proteins.

What is Aromaphilicity?

“…The aromaphilicity index is expected to be useful for understanding various phenomena occurring in proteins, peptides, and amino acids. Aromaphilicity was recently suggested to be related to the mechanism of liquid-liquid phase separation of proteins and peptides [19][20][21], which suggests the potential application of the index for describing and understanding residue-residue interactions in proteins and peptides. A for future directions includes the design of amino acid sequences in proteins and peptides as biopharmaceuticals on the basis of the aromaphilicity index.…”

The Effect of Polymer Length in Phase Separation

“…Lim et al 15 observed condensates in 23-residue peptides rich in histidine and tyrosine. Akahoshi et al 16 observed condensation with polyA 15 ssDNA and K 7 X 3 peptides, where X represents several different residues. Tang et al 17 conducted an exhaustive computational survey of all dipeptide sequences and experimentally demonstrated liquid droplets of QW.…”