ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism

A common strategy to increase aggregation resistance is through rational mutagenesis to supercharge proteins, which leads to 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 polyphos...

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Autores principales:	Jordan Bye, Kiah Murray, Robin Curtis
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	biopharmaceuticals protein aggregation protein–protein interactions ATP membraneless organelles protein self assembly Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/90b1b8cb0e9146aca17a9b93e2854053
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spelling	oai:doaj.org-article:90b1b8cb0e9146aca17a9b93e28540532021-11-25T16:50:06ZATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism10.3390/biomedicines91116462227-9059https://doaj.org/article/90b1b8cb0e9146aca17a9b93e28540532021-11-01T00:00:00Zhttps://www.mdpi.com/2227-9059/9/11/1646https://doaj.org/toc/2227-9059A common strategy to increase aggregation resistance is through rational mutagenesis to supercharge proteins, which leads to 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 polyphosphate ions, adenosine triphosphate (ATP) and tripolyphosphate (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. Monomer loss kinetic studies, combined with measurements of native state protein–protein interactions and ζ-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), α-chymotrypsinogen (α-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 α-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. We expect electrostatic interactions are a significant factor in determining the stabilizing ability of ATP towards maintaining proteins in non-dispersed states in vivo.Jordan ByeKiah MurrayRobin CurtisMDPI AGarticlebiopharmaceuticalsprotein aggregationprotein–protein interactionsATPmembraneless organellesprotein self assemblyBiology (General)QH301-705.5ENBiomedicines, Vol 9, Iss 1646, p 1646 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	biopharmaceuticals protein aggregation protein–protein interactions ATP membraneless organelles protein self assembly Biology (General) QH301-705.5
spellingShingle	biopharmaceuticals protein aggregation protein–protein interactions ATP membraneless organelles protein self assembly Biology (General) QH301-705.5 Jordan Bye Kiah Murray Robin Curtis ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism
description	A common strategy to increase aggregation resistance is through rational mutagenesis to supercharge proteins, which leads to 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 polyphosphate ions, adenosine triphosphate (ATP) and tripolyphosphate (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. Monomer loss kinetic studies, combined with measurements of native state protein–protein interactions and ζ-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), α-chymotrypsinogen (α-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 α-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. We expect electrostatic interactions are a significant factor in determining the stabilizing ability of ATP towards maintaining proteins in non-dispersed states in vivo.
format	article
author	Jordan Bye Kiah Murray Robin Curtis
author_facet	Jordan Bye Kiah Murray Robin Curtis
author_sort	Jordan Bye
title	ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism
title_short	ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism
title_full	ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism
title_fullStr	ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism
title_full_unstemmed	ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism
title_sort	atp and tri-polyphosphate (tpp) suppress protein aggregate growth by a supercharging mechanism
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/90b1b8cb0e9146aca17a9b93e2854053
work_keys_str_mv	AT jordanbye atpandtripolyphosphatetppsuppressproteinaggregategrowthbyasuperchargingmechanism AT kiahmurray atpandtripolyphosphatetppsuppressproteinaggregategrowthbyasuperchargingmechanism AT robincurtis atpandtripolyphosphatetppsuppressproteinaggregategrowthbyasuperchargingmechanism
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ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism

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