Aggregate Suppression of Recombinant Human Serum Albumin (HSA) by Adenosine Triphosphate (ATP).

Adenosine triphosphate (ATP), a key cellular energy metabolite, has been shown to modulate protein self-assembly processes such as amyloid formation and the behavior of biological condensates through nonspecific, proteome-wide mechanisms. Gaining mechanistic insight into these effects may enable the rational use of multivalent phosphate ions as stabilizing additives for biologics. The stabilizing properties of ATP are often attributed to its hydrotropic character, arising from its combination of a nonpolar adenosine moiety and a highly charged triphosphate group. However, this explanation has been questioned in cases where ATP behaves similarly to the simpler tripolyphosphate (TPP) ion. In this study, we compare the effects of ATP and TPP on the solution behavior of recombinant human serum albumin (HSA). A combination of ζ-potential measurements and saturation transfer difference (STD) NMR reveals that both ATP and TPP exhibit similar binding profiles, interacting with multiple sites on the protein with millimolar affinities. Thermal denaturation experiments indicate stronger binding to the unfolded state than to the native folded conformation, consistent with their equivalent effectiveness in suppressing aggregation under thermal stress. The stabilization effect arises from a dual mechanism: supercharging of the protein, which enhances colloidal stability, and attenuation of short-range attractive interactions. Notably, differences between ATP and TPP only emerge at elevated salt concentrations. These findings are interpreted in light of known interactions with intrinsically disordered protein regions, where ATP binds positively charged residues (particularly arginine) at submillimolar concentrations and interacts with aromatic residues at concentrations above 10 mM. Together, these results support a general framework in which multivalent anions such as ATP and TPP enhance the solubility of partially folded proteins, with implications for the design of next-generation excipients in biopharmaceutical formulations.