Bioinspired phase-separating peptides: uncovering the structural basis of liquid liquid phase separation

• Main Author: Lim, Jessica Jiaying
• Other Authors: Konstantin Pervushin
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2025
• Subjects: Chemistry; Medicine, Health and Life Sciences; Liquid liquid phase separation; Nuclear magnetic resonance (NMR)
• Online Access: https://hdl.handle.net/10356/182954
• Institution: Nanyang Technological University

Liquid liquid phase separation (LLPS) has burgeoned to be an intriguing research area with far-reaching implications in various fields. Yet, comparatively to the field’s thorough exploration of the driving forces behind LLPS, little is known about the internal structure and organization of the droplets formed. With efforts to close this gap increasing only in recent years, we are still missing a perceived linkage between what we see at the atomic and molecular level to the larger mesoscale structure. Here, using a squid-beak-derived minimal peptide system, GY23, as our key model system, we establish methods mainly using solution- and solid-state Nuclear Magnetic Resonance (NMR) to uncover the hierarchical organization of the phase-separated peptides. Key residues were identified through mutagenesis studies, and by combining NMR data with quantum chemistry calculations, atomic-level configurations of interaction nodes were predicted. Transferred Nuclear Overhauser Effect Spectroscopy (TrNOESY) then revealed the preservation of topology at the NMR timescale, responsible for associating several peptides into mesoscale clusters. Data obtained from Small Angle Neutron Scattering (SANS) and brightfield microscopy demonstrated the organization of these clusters into a granular, nanoporous structure, supported by evidence from 19F solid-state NMR of the coexistence of diffusive regions interspersed with dipolar-ordered complexes of interacting peptides. Systematic mutagenesis studies then explored the importance of the balance of interactions at the peptide level in forming the volume-spanning network while a brief exploration of heme coordination and conferred pseudo-peroxidase activity alludes to the functional consequences of the network formed. The overall work significantly advances our understanding of the dense phase, which is typically challenging to study using direct NMR and other atomic-resolution techniques. Notably, the analysis of nanoscale topologies may pave the way in linking to macroscale properties with direct implications for engineering future applications of these droplets.