Only kosmotrope anions trigger fibrillization of the recombinant core spidroin eADF4(C16) from Araneus diadematus.

Recombinant core spidroin eADF4(C16) has received increasing attention due to its ability to form micro- and nano-structured scaffolds, which are based on nanofibrils with great potential for biomedical and biotechnological applications. Phosphate anions have been demonstrated to trigger the eADF4(C16) self-assembly into cross-beta fibrils. In the present work, we systematically addressed the effect of nine sodium anions, namely SO4 2- , HPO4 2- (Pi), F- , Cl- , Br- , NO3 - , I- , SCN- , and ClO4 - from the Hofmeister series on the in vitro self-assembly kinetics of eADF4(C16). We show that besides the phosphate anions, only kosmotropic anions such as sulfate and fluoride can initiate the eADF4(C16) fibril formation. Global analysis of the self-assembly kinetics, utilizing the platform AmyloFit, showed the nucleation-based mechanism with a major role of secondary nucleation, surprisingly independent of the type of the kosmotropic anion. The rate constant of the fibril elongation in mixtures of phosphate anions with other studied anions correlated with their kosmotropic or chaotropic position in the Hofmeister series. Our findings suggest an important role of anion hydration in the eADF4(C16) fibrillization process.