The role of phosphate in silk fibroin self-assembly: a Hofmeister study

Silk fibroin is the primary protein component of the threads of Bombyx mori silkworm cocoons. Previous work has demonstrated that silk fibroin can self-assemble at solid-liquid interfaces to form dense, nanothin coatings that grow continuously from a substrate surface when exposed to potassium phosphate, a kosmotropic salt. Herein, the role of potassium phosphate in promoting silk fibroin self-assembly in solution and on surfaces is studied and compared to other salts in the Hofmeister series. Results show that strong kosmotropes, such as ammonium sulfate and potassium phosphate, promote a bimodal distribution of assembled species in solution that is indicative of a nucleation-growth mechanism. Interestingly, silk fibroin assemblies formed by potassium phosphate contain the highest β-sheet content, suggesting that phosphate-specific interactions play a role in silk fibroin self-assembly. In the presence of kosmotropic salts, silk fibroin nanoaggregates continuously accumulate at solid-liquid interfaces with varying early- and late-stage adsorption rates. Interfacial coatings formed in the presence of potassium phosphate are smooth, dense, and completely cover the underlying substrate without evidence of large-scale aggregation, whereas other kosmotropes generate rough, heterogeneous coatings. These studies thus decouple the kosmotropic effects of phosphate (via disruption of the protein hydration shell) from ion-specific behavior in driving silk fibroin self-assembly.