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Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2022,5,31]]},"abstract":"<jats:p>\n                    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 \u03b2\n                    <jats:sub>2<\/jats:sub>\n                    -microglobulin (\u03b2\n                    <jats:sub>2<\/jats:sub>\n                    m) as a model system, focusing on D76N-\u03b2\n                    <jats:sub>2<\/jats:sub>\n                    m that is involved in hereditary amyloidosis. This amino acid substitution causes the aggregation-resilient wild-type protein to become highly aggregation prone in\u00a0vitro, although the mechanism by which this occurs remained elusive. Here, we identify the residues key to protecting \u03b2\n                    <jats:sub>2<\/jats:sub>\n                    m from aggregation by coupling aggregation with antibiotic resistance in\n                    <jats:italic>E. coli<\/jats:italic>\n                    using a tripartite \u03b2-lactamase assay (TPBLA). By performing saturation mutagenesis at three different sites (D53X-, D76X-, and D98X-\u03b2\n                    <jats:sub>2<\/jats:sub>\n                    m) we show that residue 76 has a unique ability to drive \u03b2\n                    <jats:sub>2<\/jats:sub>\n                    m aggregation in\u00a0vivo and in\u00a0vitro. Using a randomly mutated D76N-\u03b2\n                    <jats:sub>2<\/jats:sub>\n                    m 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-\u03b2\n                    <jats:sub>2<\/jats:sub>\n                    m aggregation involving the formation of long-range couplings between the APR and Asn76 in a nonnative state.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2200468119","type":"journal-article","created":{"date-parts":[[2022,5,25]],"date-time":"2022-05-25T13:16:33Z","timestamp":1653484593000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":12,"title":["The effect of mutation on an aggregation-prone protein: An in\u00a0vivo, in\u00a0vitro, and in silico analysis"],"prefix":"10.1073","volume":"119","author":[{"given":"N.","family":"Guthertz","sequence":"first","affiliation":[{"name":"Astbury Centre for Structural Molecular Biology, School of Molecular &amp; Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R.","family":"van der Kant","sequence":"additional","affiliation":[{"name":"Switch Laboratory, VIB-KU Leuven Center for Brain &amp; Disease Research, 3000 Leuven, Belgium"},{"name":"Department of Cellular and Molecular Medicine, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R. M.","family":"Martinez","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural Molecular Biology, School of Molecular &amp; Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8389-4120","authenticated-orcid":false,"given":"Y.","family":"Xu","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural Molecular Biology, School of Molecular &amp; Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"C. H.","family":"Trinh","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural Molecular Biology, School of Molecular &amp; Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0392-1350","authenticated-orcid":false,"given":"B. I.","family":"Iorga","sequence":"additional","affiliation":[{"name":"Universit\u00e9 Paris-Saclay, CNRS UPR 2301, Institut de Chimie des Substances Naturelles, 91198 Gif-sur-Yvette, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"F.","family":"Rousseau","sequence":"additional","affiliation":[{"name":"Switch Laboratory, VIB-KU Leuven Center for Brain &amp; Disease Research, 3000 Leuven, Belgium"},{"name":"Department of Cellular and Molecular Medicine, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J.","family":"Schymkowitz","sequence":"additional","affiliation":[{"name":"Switch Laboratory, VIB-KU Leuven Center for Brain &amp; Disease Research, 3000 Leuven, Belgium"},{"name":"Department of Cellular and Molecular Medicine, KU Leuven, 3000 Leuven, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0802-5937","authenticated-orcid":false,"given":"D. 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E.","family":"Radford","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural Molecular Biology, School of Molecular &amp; Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2022,5,25]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-018-0060-8"},{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.csbj.2020.05.026"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2020.01.005"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2019.09.001"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.2337\/diab.45.9.1279"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1038\/nn0901-887"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41380-019-0410-8"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1136\/jnnp-2014-308724"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1038\/nbt1000"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41572-018-0034-3"},{"key":"e_1_3_4_11_2","first-page":"153","article-title":"Clearance and synthesis rates of beta 2-microglobulin in patients undergoing hemodialysis and in normal subjects","volume":"118","author":"Floege J.","year":"1991","unstructured":"J. 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Guthertz\u00a0. Dataset associated with \u201cThe effect of mutation on an aggregation-prone protein: An in vivo in vitro and in silico analysis.\u201d Dataset. https:\/\/doi.org\/10.5518\/1073. Deposited 11 November 2021."}],"container-title":["Proceedings of the National Academy of Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pnas.org\/doi\/pdf\/10.1073\/pnas.2200468119","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,1,23]],"date-time":"2023-01-23T16:09:35Z","timestamp":1674490175000},"score":1,"resource":{"primary":{"URL":"https:\/\/pnas.org\/doi\/10.1073\/pnas.2200468119"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,5,25]]},"references-count":62,"journal-issue":{"issue":"22","published-print":{"date-parts":[[2022,5,31]]}},"alternative-id":["10.1073\/pnas.2200468119"],"URL":"https:\/\/doi.org\/10.1073\/pnas.2200468119","relation":{},"ISSN":["0027-8424","1091-6490"],"issn-type":[{"value":"0027-8424","type":"print"},{"value":"1091-6490","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,5,25]]},"assertion":[{"value":"2022-01-10","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2022-04-13","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2022-05-25","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e2200468119"}}