{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T22:29:13Z","timestamp":1787005753184,"version":"build-2736575974"},"reference-count":64,"publisher":"National Academy of Sciences","issue":"38","license":[{"start":{"date-parts":[[2025,9,17]],"date-time":"2025-09-17T00:00:00Z","timestamp":1758067200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["104918MA"],"award-info":[{"award-number":["104918MA"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000268","name":"UKRI | Biotechnology and Biological Sciences Research Council","doi-asserted-by":"publisher","award":["BB\/W510403\/1"],"award-info":[{"award-number":["BB\/W510403\/1"]}],"id":[{"id":"10.13039\/501100000268","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000268","name":"UKRI | Biotechnology and Biological Sciences Research Council","doi-asserted-by":"publisher","award":["BB\/T007222\/1"],"award-info":[{"award-number":["BB\/T007222\/1"]}],"id":[{"id":"10.13039\/501100000268","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000288","name":"Royal Society","doi-asserted-by":"publisher","award":["RSRP\/R1\/211057"],"award-info":[{"award-number":["RSRP\/R1\/211057"]}],"id":[{"id":"10.13039\/501100000288","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2025,9,23]]},"abstract":"<jats:p>\n                    Deep mutational scanning (DMS) assays provide a powerful method to generate large-scale datasets essential for advancing AI-driven predictions in biology. The tripartite \u03b2-lactamase assay (TPBLA), in which a protein of interest is inserted between two domains of \u03b2-lactamase, has previously been reported as capable of detecting and quantitating the aggregation of proteins and biologics in the oxidizing periplasm of\n                    <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                    and used as a platform for identifying small molecule inhibitors of aggregation. Here, we repurpose the TPBLA into a high-throughput DMS platform. We validate this format using a single-site saturation library of the intrinsically disordered peptide A\u03b2\n                    <jats:sub>42<\/jats:sub>\n                    , linked to Alzheimer\u2019s disease, demonstrating strong agreement between observed variant fitness scores and variant behavior using our previously reported low-throughput TPBLA. The results of DMS revealed variant fitness scores that correlate with known amyloid-promoting regions. An in silico approach using FoldX-derived per-residue thermodynamic stability confirmed that the TPBLA reports on amyloid fibril stability. In vitro experiments support this finding, showing a strong correlation between variant fitness scores and the critical concentration of amyloid formation. Machine learning using the DMS dataset identified \u03b2\u2010sheet propensity and polarity as primary drivers of variant fitness scores. The derived model is also able to predict thermodynamically stabilizing regions in other amyloid systems, underscoring its generalizability. Collectively, our results demonstrate the TPBLA as a versatile platform for generating robust datasets to advance predictive modeling and to inform the design of aggregation\u2010resistant proteins.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2516165122","type":"journal-article","created":{"date-parts":[[2025,9,17]],"date-time":"2025-09-17T17:47:38Z","timestamp":1758131258000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":4,"title":["Employing deep mutational scanning in the\n                    <i>Escherichia coli<\/i>\n                    periplasm to decode the thermodynamic landscape for amyloid formation"],"prefix":"10.1073","volume":"122","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-7357-5441","authenticated-orcid":false,"given":"Conor E.","family":"McKay","sequence":"first","affiliation":[{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miles","family":"Deans","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jack","family":"Connor","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Janet C.","family":"Saunders","sequence":"additional","affiliation":[{"name":"The Discovery Centre, AstraZeneca","place":["Cambridge, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher","family":"Lloyd","sequence":"additional","affiliation":[{"name":"The Discovery Centre, AstraZeneca","place":["Cambridge, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sheena E.","family":"Radford","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0802-5937","authenticated-orcid":false,"given":"David J.","family":"Brockwell","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2025,9,17]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2836(77)80200-3"},{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03819-2"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-07487-w"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btm098"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.ads0018"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.79932"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41588-019-0431-x"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.3389\/fgene.2023.1087267"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41436-021-01223-9"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-024-03444-y"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-15667-1"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.1988"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2009.11.022"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2200468119"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.trechm.2022.02.004"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.63364"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-53195-4"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-07966-0"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-35940-3"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2026658118"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2003.11.008"},{"key":"e_1_3_4_22_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.274.18.12619"},{"key":"e_1_3_4_23_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2014.09.026"},{"key":"e_1_3_4_24_2","doi-asserted-by":"publisher","DOI":"10.1038\/nbt1012"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-017-13210-9"},{"key":"e_1_3_4_26_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-8-65"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.1336-09.2009"},{"key":"e_1_3_4_28_2","doi-asserted-by":"publisher","DOI":"10.15252\/embj.2019102864"},{"key":"e_1_3_4_29_2","doi-asserted-by":"publisher","DOI":"10.1038\/282109a0"},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41380-021-01249-0"},{"key":"e_1_3_4_31_2","doi-asserted-by":"publisher","DOI":"10.1002\/biot.201400291"},{"key":"e_1_3_4_32_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-0524-0_38"},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1534\/g3.119.400535"},{"key":"e_1_3_4_34_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-34742-3"},{"key":"e_1_3_4_35_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2021.08.013"},{"key":"e_1_3_4_36_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gki387"},{"key":"e_1_3_4_37_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2022.05.002"},{"key":"e_1_3_4_38_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2025.07.005"},{"key":"e_1_3_4_39_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41594-024-01252-3"},{"key":"e_1_3_4_40_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.aao2825"},{"key":"e_1_3_4_41_2","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.6b03715"},{"key":"e_1_3_4_42_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41593-023-01484-4"},{"key":"e_1_3_4_43_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpc.2021.106549"},{"key":"e_1_3_4_44_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-19961-3"},{"key":"e_1_3_4_45_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.abb.2024.110179"},{"key":"e_1_3_4_46_2","doi-asserted-by":"publisher","DOI":"10.1098\/rsos.160696"},{"key":"e_1_3_4_47_2","doi-asserted-by":"publisher","DOI":"10.1063\/1.5133635"},{"key":"e_1_3_4_48_2","unstructured":"A. Vaswani Attention is all you need. arXiv [Preprint] (2017). https:\/\/doi.org\/10.48550\/arXiv.1706.03762 (Accessed 3 September 2025)."},{"key":"e_1_3_4_49_2","doi-asserted-by":"publisher","DOI":"10.1385\/1592598900"},{"key":"e_1_3_4_50_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bty140"},{"key":"e_1_3_4_51_2","doi-asserted-by":"publisher","DOI":"10.1111\/cts.70056"},{"key":"e_1_3_4_52_2","doi-asserted-by":"publisher","DOI":"10.1002\/cpt.3076"},{"key":"e_1_3_4_53_2","doi-asserted-by":"publisher","DOI":"10.1002\/psp4.12796"},{"key":"e_1_3_4_54_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-44489-0"},{"key":"e_1_3_4_55_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41593-023-01341-4"},{"key":"e_1_3_4_56_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-04199-3"},{"key":"e_1_3_4_57_2","doi-asserted-by":"crossref","unstructured":"A. Arutyunyan M. Seuma A. J. Faure B. Bolognesi B. Lehner Massively parallel genetic perturbation suggests the energetic structure of an amyloid-\u03b2 transition state. Sci. Adv. 11 eadv1422 (2025).","DOI":"10.1126\/sciadv.adv1422"},{"key":"e_1_3_4_58_2","doi-asserted-by":"publisher","DOI":"10.1002\/ana.21321"},{"key":"e_1_3_4_59_2","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.4c16743"},{"key":"e_1_3_4_60_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-28660-7"},{"key":"e_1_3_4_61_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41572-018-0034-3"},{"key":"e_1_3_4_62_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-06761-6"},{"key":"e_1_3_4_63_2","doi-asserted-by":"publisher","DOI":"10.1128\/MMBR.67.4.593-656.2003"},{"key":"e_1_3_4_64_2","unstructured":"C. McKay Data from \u201cEmploying deep mutational scanning in the Escherichia coli periplasm to decode the thermodynamic landscape for amyloid formation.\u201d Research Data Leeds Repository. https:\/\/doi.org\/10.5518\/1653. Deposited 26 June 2025."}],"container-title":["Proceedings of the National Academy of Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pnas.org\/doi\/pdf\/10.1073\/pnas.2516165122","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,9,17]],"date-time":"2025-09-17T17:47:44Z","timestamp":1758131264000},"score":1,"resource":{"primary":{"URL":"https:\/\/pnas.org\/doi\/10.1073\/pnas.2516165122"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,17]]},"references-count":64,"journal-issue":{"issue":"38","published-print":{"date-parts":[[2025,9,23]]}},"alternative-id":["10.1073\/pnas.2516165122"],"URL":"https:\/\/doi.org\/10.1073\/pnas.2516165122","relation":{},"ISSN":["0027-8424","1091-6490"],"issn-type":[{"value":"0027-8424","type":"print"},{"value":"1091-6490","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,9,17]]},"assertion":[{"value":"2025-06-20","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-08-11","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-09-17","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e2516165122"}}