A Molecular view on Protein - Ion Interactions during the Fibrinogen Aggregate Formation†

Understanding how ions regulate fibrinogen self-assembly into fibers is essential for advancing enzymefree strategies to fabricate fibrillar biomaterials. Here, we combine evaporation-driven self-assembly experiments with atomistic molecular dynamics simulations to elucidate how chloride-and phosphatebased salt environments modulate the nanoscale organization and electrostatic landscape of fibrinogen. Mass-loss and turbidity data identified a critical salt threshold above which rapid fiber growth occured. SEM-EDS analysis further showed selective retention of sodium, particularly associated with phosphate, while chloride was largely removed, indicating different and non-equivalent roles of the mixed-salts in the assembly of fibrinogen nanofibers during the drying process. Simulations of the fibrinogen D domain (Fg-D) show that Na + ions form a tightly bound Stern layer at acidic residues, whereas Cloccupies the diffuse layer and exhibits only transient interactions. In contrast, monohydrogen and dihydrogen phosphate anions display strong and persistent binding to positively charged patches. The ability to be chelated by more than one positive amino acid side chain and fixation/clustering of immobilized Na + ions at Ca 2+-binding sites lead to reduced ion mobility, extended hydration structuring, and pronounced modulation-or even inversion-of the local electrostatic potential. These ion-specific effects collectively lower the electrostatic barrier for protein-protein approach and explain the strong aggregation propensity observed experimentally in phosphate-rich environments. By revealing how distinct ions tune the electrical double layer and solvent structure around Fg-D, this work establishes a physicochemical basis for designing enzyme-free fibrinogen materials with controllable fibrillar architecture and offers new opportunities for biomaterial development using tailored ionic environments. † Electronic supplementary information (ESI) available.