Length-encoded phase transitions in proline-alanine-serine peptides: from nanoaggregates to condensates.

PAS peptides form nanoscale aggregates and only the longest chains undergo significant phase separation, which could have implications for drug delivery systems.

Summary and findings

This study investigates the aggregation and phase separation behavior of PAS-based polypeptides composed of Proline, Alanine, and Serine residues. The research found that PAS peptides form nanoscale aggregates and that only the longest PAS80 chains undergo liquid-liquid phase separation. The study utilized spectroscopy, microscopy, and molecular dynamics simulations to analyze these behaviors.

Abstract

PAS-based polypeptides, composed of uncharged Proline, Alanine, and Serine residues, are intrinsically disordered polymers used to enhance biologics' solubility and pharmacokinetics. Despite their biomedical relevance, their aggregation and phase separation behaviour remain underexplored. Here we combined spectroscopy, microscopy and molecular dynamics simulations to investigate the structure of PAS peptides of varying lengths (20-80 residues). We found that all peptides form nanoscale aggregates (50-350 nm) above their critical concentrations, yet only the longest PAS80 chains undergo liquid-liquid phase separation, yielding micrometre-scale condensates over a broad concentration range (0.15-6 mM), as revealed by the phase diagram. Monitoring the phase-separation process over time provided direct evidence that condensates undergo fusion with droplet sizes increasing from approximately 1 μm to 5 μm. Fluorescence recovery after photobleaching (FRAP) microscopy and nuclear magnetic resonance (NMR) spectroscopy revealed that PAS80 condensates retain a high degree of molecular mobility and exhibit characteristic liquid-like behaviour. Furthermore, treatment of the condensates with a hydrogen-bond-disrupting agent, an amphiphilic solvent, and heating demonstrated that weak interactions between hydrophobic amino acids and the entropic gain associated with the hydrophobic effect are the primary driving forces underlying the formation of condensates. In contrast, hydrogen bonding was found to play a secondary role, likely contributing to the further stabilization of the assembled structures. Molecular dynamics simulations of isolated and clustered PAS chains revealed that individual PAS peptides mainly exist as highly flexible but compact random-coil structures, and that the transition from extended to compact conformations is driven by entropic effects. Furthermore, PAS peptides rapidly self-assemble into clusters that maintain random-coil conformations and display substantial structural flexibility and configurational heterogeneity. These findings elucidate the molecular basis of PAS peptides phase behaviour and establish PAS peptides as promising building blocks for the rational design of bioengineered coacervates with potential applications in drug delivery and compartmentalized biocatalysis.

Key findings

• Peptides form nanoscale aggregates (50-350 nm) above critical concentrations.
• Only PAS80 chains undergo liquid-liquid phase separation, yielding micrometre-scale condensates over a concentration range of 0.15-6 mM.
• Condensate droplet sizes increase from approximately 1 μm to 5 μm during fusion.
• Hydrophobic interactions and entropic gain are primary driving forces for condensate formation.
• Individual PAS peptides exist as highly flexible but compact random-coil structures.

Limitations

• Not reported in abstract.
• No human data provided.
• Study primarily focused on in vitro conditions.
• Potential confounding factors related to experimental design.

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