# Stepwise LCST‐Type Phase Separation in Mixtures of Short‐Chain Elastin‐Like Peptides With Minimal Structural Differences

## TL;DR

Short-chain elastin-like peptides with minor structural differences show stepwise phase separation when heated, offering new ways to design responsive materials.

## Contribution

Demonstrates that small chain-length variations in ELPs can lead to distinct phase behaviors in mixtures.

## Key findings

- Turbidity and UPLC-MS showed stepwise phase transitions in mixed ELP solutions upon heating.
- Structural transitions from polyproline type II helix to β-sheet or β-turn were observed.
- Heterotypic interactions influence the sequential phase behavior in ELP mixtures.

## Abstract

Elastin‐like peptides (ELPs) comprise repetitive pentapeptide sequences and exhibit liquid–liquid phase separation through lower critical solution temperature‐type behavior. Their stimuli‐responsive behavior has enabled diverse applications in biomedical and chemical contexts. Although the miscibility and interactions of ELP mixtures have been previously studied, it remains unclear whether mixtures of short‐chain ELPs with minimal differences in intrinsic parameters, such as chain length, can exhibit distinct phase behaviors. In this study, we investigated whether synthetic short‐chain ELPs differing in length by only one or two repeat units (i.e., 5 or 10 residues) could exhibit independent phase transitions in mixed systems. Turbidity measurements of single‐ and two‐component ELP solutions supported by UPLC‐MS analysis revealed stepwise phase transitions upon heating. Our mechanistic analyses revealed that the mixtures undergo a structural transition from polyproline type II helix to β‐sheet or β‐turn structures. In addition, although the mixtures exhibited stepwise phase separation, our results indicate that heterotypic interactions influenced the sequential phase behavior. These findings reveal that even subtle variations in the ELP chain length and architecture can drive distinct phase separation, providing a rational strategy for designing functional, multicomponent, responsive peptide‐based materials.

Small differences in chain‐length or topology induce stepwise phase separation in short‐chain ELP mixtures.