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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| Format: | Article |
| Language: | English |
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MDPI AG
2021-11-01
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| Series: | Biomedicines |
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| Online Access: | https://www.mdpi.com/2227-9059/9/11/1646 |
| _version_ | 1797511153520214016 |
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| author | Jordan Bye Kiah Murray Robin Curtis |
| author_facet | Jordan Bye Kiah Murray Robin Curtis |
| author_sort | Jordan Bye |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T05:41:24Z |
| format | Article |
| id | doaj.art-90b1b8cb0e9146aca17a9b93e2854053 |
| institution | Directory Open Access Journal |
| issn | 2227-9059 |
| language | English |
| last_indexed | 2024-03-10T05:41:24Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomedicines |
| spelling | doaj.art-90b1b8cb0e9146aca17a9b93e28540532023-11-22T22:31:19ZengMDPI AGBiomedicines2227-90592021-11-01911164610.3390/biomedicines9111646ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging MechanismJordan Bye0Kiah Murray1Robin Curtis2Department of Chemical Engineering and Analytical Science, University of Manchester, Manchester M1 7DN, UKDepartment of Chemical Engineering and Analytical Science, University of Manchester, Manchester M1 7DN, UKDepartment of Chemical Engineering and Analytical Science, University of Manchester, Manchester M1 7DN, UKA 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.https://www.mdpi.com/2227-9059/9/11/1646biopharmaceuticalsprotein aggregationprotein–protein interactionsATPmembraneless organellesprotein self assembly |
| spellingShingle | Jordan Bye Kiah Murray Robin Curtis ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism Biomedicines biopharmaceuticals protein aggregation protein–protein interactions ATP membraneless organelles protein self assembly |
| title | 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_short | 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 |
| topic | biopharmaceuticals protein aggregation protein–protein interactions ATP membraneless organelles protein self assembly |
| url | https://www.mdpi.com/2227-9059/9/11/1646 |
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