Peptide-based coacervates have emerged as versatile platforms for drug delivery, tissue scaffolding, bioreactor systems, and cellular regulation due to their sequence programmability, dynamic self-assembly, and exceptional biocompatibility. These liquid-liquid phase-separated droplets form through complex intermolecular interactions including electrostatic forces, hydrophobic contacts, hydrogen bonds, and cation-π interactions. However, this complexity impedes precise control over microenvironmental properties and obscures the connections between peptide sequence, phase behavior, and biological function. Establishing clear structure-function relationships requires systematic examination of how specific amino acid substitutions modulate the underlying molecular interactions and resulting condensate properties.
Researchers in the Tian Group at Zhejiang University, Hangzhou, China, published in JACS, investigated how single amino acid substitutions in decapeptide sequences alter coacervate microenvironments and biological functions. The team constructed model systems using decaarginine, decalysine, and decaaspartic acid, then systematically replaced aspartic acid with phenylalanine or lysine with arginine to examine hydrophobic and electrostatic contributions respectively. Increasing the phenylalanine percentage in the R10/D10 system dramatically enhanced phase separation propensity, reducing the minimum phase-separating peptide concentration from 0.25 to 0.01 mM while elevating the critical salt concentration from 150 to 350 mM. Raman spectroscopy confirmed improved hydrophobic packing through characteristic blue shifts in the amide III band. Fluorescence lifetime imaging revealed that internal polarity decreased substantially as phenylalanine content rose, with probe lifetimes increasing from 1.8 to 5.2 nanoseconds. Fluorescence recovery after photobleaching measurements showed corresponding reductions in molecular mobility, with diffusion coefficients dropping from 5.6 × 10−4 to 0.86 × 10−4 μm2/s.
Isothermal titration calorimetry uncovered a striking thermodynamic transition. At low phenylalanine percentages, enthalpy dominated the driving force for phase separation, with ΔH values around −30 kJ/mol. As phenylalanine content increased, the system shifted to entropy-driven assembly with ΔH stabilizing near −0.5 kJ/mol, reflecting enhanced dehydration effects from hydrophobic interactions. In contrast, substituting lysine with arginine in the K10/D10 system produced more modest effects. Stable droplets formed only when arginine exceeded 20%, and while salt resistance and viscosity increased with arginine content, the changes remained substantially smaller than those achieved through phenylalanine substitution. Neither substitution altered the enrichment of biomacromolecules such as single-stranded DNA, horseradish peroxidase, or bovine serum albumin. However, phenylalanine substitution profoundly affected nucleic acid structure within droplets. Using FRET-based fluorescence lifetime imaging with a 40-base pair DNA probe, the researchers demonstrated that increasing phenylalanine content progressively enhanced double-stranded DNA unwinding, with FRET efficiency dropping from 0.70 to 0.20 as phenylalanine reached 50%. Arginine substitution showed no such effect, revealing that hydrophobic microenvironments specifically modulate nucleic acid secondary structure.
Droplet mixing experiments demonstrated that high phenylalanine content created strong interfacial barriers preventing material exchange between droplets, while arginine-rich systems maintained molecular mobility allowing DNA probes to diffuse and hybridize across droplet boundaries. Cell coculture experiments with 293T cells showed that both R10/(FD)5 and R10/D10 coacervates adhered rapidly to cell membranes within 15 minutes while maintaining greater than 94% cell viability. However, R10/D10 inhibited proliferation by 20% compared to 10% for R10/(FD)5. Transcriptome analysis revealed distinct pathway alterations: R10/D10 affected cell cycle and MAPK signaling while R10/(FD)5 influenced DNA damage checkpoints and glucose metabolism. Western blot confirmed downregulation of the proliferation-related protein MAPK11 in both systems. Confocal microscopy indicated that coacervates remained membrane-associated rather than internalized, suggesting that gene expression changes arise from extracellular interactions rather than intracellular signaling. This molecular-level analysis establishes foundations for designing functional condensates capable of modulating cellular fate through programmable microenvironments.
