ATP Can Act as a Stabilizer on Neutral Macromolecules

Adenosine triphosphate (ATP), an important biomolecule, plays a vital role in delivering cellular energy for various bioprocesses. It was recently shown that ATP also serves as a hydrotrope, destabilizing protein coacervates. Herein, we studied the influence of ATP and relevant small molecules (adenine, adenosine, adenosine monophosphate (AMP), and triphosphate (TP)) on the phase transition of macromolecules, i.e., poly(N-isopropylacrylamide), to explore the underlying mechanism of hydrotropic action of ATP. A multi-instrumental approach, utilizing the Lower Critical Solution Temperature (LCST), Hydrogen-Nuclear Magnetic Resonance (¹H NMR), and ATR Fourier-Transform Infrared (ATR-FTIR), solvation shell spectroscopy, along with all-atom molecular dynamics simulations were adopted. Adenine and adenosine show a negligible effect on the solubility of macromolecules, whereas ATP, AMP, and triphosphate exhibited dominant salting-out behavior, and promoted the aggregation of neutral macromolecules. ATR-FTIR measurements support the salting-out behavior at physiological ATP concentrations (<0.1 M). In line with this, no apparent evidence for specific binding interaction between the macromolecule and ATP was observed in spectroscopic measurements, as well as MD simulations. At elevated concentrations, ATP self-associates into small clusters, resulting in the destabilization of the PNIPAM chain in its collapsed state. Overall, we demonstrate that only the presence of disordered neutral macromolecules, rich in valine-like pendant isopropyl group, are not sufficient for effective hydrotropic action of ATP; rather, ATP can stabilize such macromolecules with an excluded volume effect at physiological concentrations.