Guthertz, N orcid.org/0000-0001-7247-1176, van der Kant, R orcid.org/0000-0003-3639-9749, Martinez, RM orcid.org/0000-0002-7371-8051 et al. (7 more authors) (2022) The effect of mutation on an aggregation-prone protein: An in vivo, in vitro, and in silico analysis. Proceedings of the National Academy of Sciences (PNAS), 119 (22). e2200468119. ISSN 0027-8424
Abstract
Aggregation of initially stably structured proteins is involved in more than 20 human amyloid diseases. Despite intense research, however, how this class of proteins assembles into amyloid fibrils remains poorly understood, principally because of the complex effects of amino acid substitutions on protein stability, solubility, and aggregation propensity. We address this question using β₂-microglobulin (β₂m) as a model system, focusing on D76N-β₂m that is involved in hereditary amyloidosis. This amino acid substitution causes the aggregation-resilient wild-type protein to become highly aggregation prone in vitro, although the mechanism by which this occurs remained elusive. Here, we identify the residues key to protecting β₂m from aggregation by coupling aggregation with antibiotic resistance in E. coli using a tripartite β-lactamase assay (TPBLA). By performing saturation mutagenesis at three different sites (D53X-, D76X-, and D98X-β₂m) we show that residue 76 has a unique ability to drive β₂m aggregation in vivo and in vitro. Using a randomly mutated D76N-β₂m variant library, we show that all of the mutations found to improve protein behavior involve residues in a single aggregation-prone region (APR) (residues 60 to 66). Surprisingly, no correlation was found between protein stability and protein aggregation rate or yield, with several mutations in the APR decreasing aggregation without affecting stability. Together, the results demonstrate the power of the TPBLA to develop proteins that are resilient to aggregation and suggest a model for D76N-β₂m aggregation involving the formation of long-range couplings between the APR and Asn76 in a nonnative state.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2022 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY). |
Keywords: | Amyloid; Aggregation; Evolution |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Structural Molecular Biology (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Synthetic Biology (Leed) |
Funding Information: | Funder Grant number Wellcome Trust 204963/Z/16/Z BBSRC (Biotechnology & Biological Sciences Research Council) BB/M012573/1 Wellcome Trust 094232/Z/10/Z Wellcome Trust 094232/Z/10/Z |
Depositing User: | Symplectic Publications |
Date Deposited: | 04 May 2022 13:27 |
Last Modified: | 15 Jan 2025 11:29 |
Status: | Published |
Publisher: | National Academy of Sciences |
Identification Number: | 10.1073/pnas.2200468119 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:186372 |