Phosphate ions reverse the phase composition of silk fibroin condensates

Liquid–liquid phase separation (LLPS) is a fundamental principle in protein assembly, directing processes ranging from the formation of membraneless organelles in biological cells to the self-assembly of silk proteins. Silk LLPS is typically studied in phosphate buffers, but it can also occur spontaneously without solvent modification. By directly comparing these two pathways, we demonstrate that phosphate ions trigger assemblies fundamentally distinct from natural, salt-free LLPS. The natural process, driven by silk's heterogeneous and amphiphilic character, yields dynamic, easily disrupted condensates. In contrast, phosphate ions promote interactions that generate larger, denser, and far more stable condensates that persist after drying. We also show that fibroin distributes throughout the outer phase in natural LLPS but migrates exclusively to the dense phase with phosphate ions. This establishes that phosphate ions actively reshape both the composition and properties of silk condensates and should be wielded with care. Our insights provide critical clues into how metal ions guide silk solidification in vivo and will inform the design of improved regenerated silk feedstocks for spinning. More broadly, this work advances our understanding of protein self-assembly, with potential implications for counteracting pathological protein aggregation in disease.