ATP and tri-polyphosphate (TPP) suppress protein aggregate growth by a supercharging mechanism

Jordan Bye, Kiah Murray, Robin Curtis

Research output: Contribution to journalArticlepeer-review

Abstract

A common strategy to increase aggregation resistance is through rational mutagenesis to supercharge proteins, which leads to a high colloidal stability, but often has the undesirable effect of lowering conformational stability. We show this trade-off can be overcome by using small multivalent poly-phosphate ions, adenosine tri-phosphate (ATP) and tri-polyphosphate (TPP) as excipients. These ions are equally effective at suppressing aggregation of ovalbumin and bovine serum albumin (BSA) upon thermal stress as monitored by dynamic and static light scattering. Studies of monomer loss kinetic studies, combined with measurements of native state protein-protein interactions and z-potentials, indicate the ions reduce aggregate growth by increasing the protein colloidal stability through binding and overcharging the protein. Out of three additional proteins studied, ribonuclease A (RNaseA), a-chymotrypsinogen (a-Cgn), and lysozyme, we only observed a reduction in aggregate growth for RNaseA, although overcharging by the poly-phosphate ions still occurs for lysozyme and a-Cgn. Because the salts do not alter protein conformational stability, using them as excipients could be a promising strategy for stabilizing biopharmaceuticals once the protein structural factors that determine whether multivalent ion binding will increase colloidal stability are better elucidated. Our findings also have biological implications. Recently, it has been proposed that ATP also plays an important role in maintaining intracellular biological condensates and preventing protein aggregation in densely-packed cellular environments. As such, we expect electrostatic interactions are a significant factor in determining the stabilizing ability of ATP towards maintaining proteins in non-dispersed states in vivo.
Original languageEnglish
JournalBiomedicines
Early online date9 Nov 2021
DOIs
Publication statusPublished - 9 Nov 2021

Fingerprint

Dive into the research topics of 'ATP and tri-polyphosphate (TPP) suppress protein aggregate growth by a supercharging mechanism'. Together they form a unique fingerprint.

Cite this