Anions, Not Cations, Drive Silk Stability and Self-Assembly: Insights from Regenerated Undegummed Silk

The self-assembly of β-sheet rich proteins such as silk are triggered by physiochemical factors such as salts, pH, and flow. Anions, in particular, have been shown to significantly influence protein self-assembly in amyloids and spider silk; however, their effect on silkworm silk has not yet been investigated. This study evaluated the influence of metal ions on silk gelation kinetics by utilizing a recently developed system to dissolve undegummed Bombyx mori cocoons. While the influence of cations agreed with previous observations, with divalent cations delaying gelation more than monovalent ions, we report that anions are more influential, with phosphate and sulfate anions accelerating gelation, regardless of the valency of the associated cation, while bromide was found to delay it. Interestingly we discovered chloride stabilized silk regardless of the cation. Using these results, a linear relationship was established between gelation time and apparent dynamic hydration number, a quantitative measure of salt-water interactions. This relationship more accurately predicted silk behavior than the traditionally used Hofmeister series. Thus, it was possible to accurately predict gelation behavior for any salt for which this number has been calculated, allowing for better designs of systems that require fine control of gelation such as wet-spinning and 3D printing. This work demonstrates the significance of anions in controlling silkworm silk stability, expanding upon the currently perceived mechanism for self-assembly.