Adenosine triphosphate (ATP) is amphiphilic in nature and has the characteristics of a hydrotrope because of the charged triphosphate moiety and the large aromatic ring located on each end of its structure. Previous studies revealed that ATP can effectively maintain the solubility and prevent liquid-liquid phase separation (LLPS) of some biological proteins. In this study, we assessed the impact of ATP on the stability of a model therapeutic IgG1 antibody (MA1) to evaluate its potential application in protein formulation design. In our system, ATP promotes rapid LLPS of MA1 and we demonstrate that the ATP-MA1 static interaction drives phase separation of MA1. The attractive protein-protein interaction increased exclusively in the presence of ATP but not in the presence of other ATP analogues, such as adenosine diphosphate, adenosine monophosphate, and adenine. Through an intrinsic fluorescence quenching study, we revealed that ATP bound to MA1 electrostatically and formed static interactions; furthermore, such static ATP-MA1 interactions significantly altered the surface property of the protein and the protein-protein interactions and subsequently induced LLPS of MA1. This ATP-induced LLPS could be effectively eliminated by Mg2+, which chelated with ATP and thus negated ATP-MA1 static interaction. Our results revealed the unique molecular mechanism of ATP-induced rapid LLPS of MA1.
Keywords: ATP; electrostatic interactions; fluorescence quenching; liquid−liquid phase separation; monoclonal antibody.