Beyond classical electrostatic screening: Non-specific binding of adenosine triphosphate drives long-range protein...

Overcharging of proteins by multivalent phosphate anions, such as adenosine triphosphate (ATP) and tripolyphosphate (TPP), can strongly modulate protein-protein interactions and colloidal stability, yet its physical origin and consequences remain incompletely understood. Here we examine how ATP and sodium tripolyphosphate (STPP) regulate native-state interactions of recombinant human serum albumin (rHSA) by combining electrophoretic ζ-potential measurements, static light scattering (SLS), small-angle X-ray scattering (SAXS), and coarse-grained molecular simulations. Analysis of ζ-potential data using Poisson-Boltzmann theory indicates weak, non-specific association of multivalent phosphates at the protein interface, leading to an increase in effective surface charge with ionic strength. Consistent with this picture, SLS measurements show that the osmotic second virial coefficient (B₂₂) becomes substantially more positive in ATP- and STPP-containing solutions than in NaCl at comparable ionic strength, reflecting enhanced net repulsion. However, SAXS reveals that this repulsion is not only stronger but also effectively longer-ranged than predicted by Debye-Hückel screening based on the bulk electrolyte, demonstrating a clear breakdown of classical DLVO descriptions. Molecular simulations show that ion-ion correlations alone cannot account for these effects at protein-relevant surface charge densities; instead, weak anion binding drives overcharging and reorganises the diffuse layer through co-ion exclusion and counterion enrichment, producing an effective underscreening and a non-DLVO repulsive interaction. Together, these results establish that multivalent phosphate ions act as active regulators of protein interactions by coupling weak phosphate association to double-layer restructuring, providing a mechanistic basis for their ability to enhance colloidal stability and offering new routes for controlling aggregation in biopharmaceutical formulations.