{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T18:39:51Z","timestamp":1792607991234,"version":"4.5.0"},"reference-count":24,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T00:00:00Z","timestamp":1792540800000},"content-version":"vor","delay-in-days":20,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"},{"start":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T00:00:00Z","timestamp":1790812800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    The foot domains of polymerases from the\n                    <jats:italic>Paramyxoviridae<\/jats:italic>\n                    family mediate attachment of the viral polymerases to their nucleocapsid templates during viral RNA synthesis. Detailed structural and biophysical characterization of these foot domains reveals that they share a common structure, but when not bound to the nucleocapsid they may exist as intrinsically unstructured chains, molten globules, or highly stable folded domains. With hundreds of paramyxovirus sequences now known, the diverse family of foot domains provides a valuable system to investigate the physical and evolutionary processes that dictate protein stability. To enable rapid and robust evaluation of the relative stability of these domains, we tested the applicability of the split \u03b2\u2010lactamase assay. This assay couples \u03b2\u2010lactamase activity to the folding of a protein inserted into a loop on the enzyme, with antibiotic resistance being conferred in proportion to the stability of the insert. In this work, five foot domains of broadly varying stability were tested with this assay. We constructed a bespoke vector to facilitate the use of this assay, and we introduced a novel statistical approach that generates a probability\u2010based ranking of relative stability from the experimental data. We find that this assay reproduces the order of stabilities obtained through biophysical approaches. Though classical definitions of stability, evaluated using traditional biophysical techniques, are ill\u2010defined for proteins of low intrinsic stability, this work finds that the split \u03b2\u2010lactamase assay can be fruitfully adapted in such cases, and can be made more convenient and interpreted more rigorously through the innovations we describe. \u00a9 2026 The Author(s).\n                    <jats:italic>Current Protocols<\/jats:italic>\n                    published by Wiley Periodicals LLC.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 1<\/jats:bold>\n                    : Preparation of screening vector suBLA for ligation\u2010independent cloning\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 2<\/jats:bold>\n                    : Ligation\u2010independent cloning of inserts into screening vector suBLA\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 3<\/jats:bold>\n                    : Use of PCR to check for inserts\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 4<\/jats:bold>\n                    : Dilution series assay\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 5<\/jats:bold>\n                    : Scoring of dilution assay\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 6<\/jats:bold>\n                    : Statistical analysis of dilution assay\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70479","type":"journal-article","created":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T16:30:28Z","timestamp":1792600228000},"source":"Crossref","is-referenced-by-count":0,"title":["A Rapid Method to Rank Proteins of Widely Varying Stability: Application to Paramyxovirus Foot Domains"],"prefix":"10.1002","volume":"6","author":[{"given":"Andrew C.","family":"Hausrath","sequence":"first","affiliation":[{"name":"Institute for Society and Genetics University of California, Los Angeles  Los Angeles California"},{"name":"Department of Microbiology, Immunology, and Molecular Genetics University of California, Los Angeles  Los Angeles California"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard L.","family":"Kingston","sequence":"additional","affiliation":[{"name":"School of Biological Sciences University of Auckland  Auckland New Zealand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Megan M.","family":"McEvoy","sequence":"additional","affiliation":[{"name":"Institute for Society and Genetics University of California, Los Angeles  Los Angeles California"},{"name":"Department of Microbiology, Immunology, and Molecular Genetics University of California, Los Angeles  Los Angeles California"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,10,21]]},"reference":[{"key":"e_1_2_12_2_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/18.20.6069"},{"key":"e_1_2_12_3_1","unstructured":"Burgess\u2010Brown N.(2013). pLIC\u2010SGC1 [Data set]. Addgene."},{"key":"e_1_2_12_4_1","doi-asserted-by":"publisher","DOI":"10.2307\/3001491"},{"key":"e_1_2_12_5_1","first-page":"403","volume-title":"Advances in Virus Research","author":"Curran J.","year":"1999"},{"key":"e_1_2_12_6_1","doi-asserted-by":"publisher","DOI":"10.1002\/9780471729259.mca02as00"},{"key":"e_1_2_12_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2009.11.022"},{"key":"e_1_2_12_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.200390046"},{"key":"e_1_2_12_9_1","doi-asserted-by":"publisher","DOI":"10.1128\/jb.164.1.70\u201077.1985"},{"key":"e_1_2_12_10_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00018\u2010017\u20102558\u20101"},{"key":"e_1_2_12_11_1","doi-asserted-by":"publisher","DOI":"10.1002\/prot.340160406"},{"key":"e_1_2_12_12_1","doi-asserted-by":"publisher","DOI":"10.1093\/protein\/14.6.415"},{"key":"e_1_2_12_13_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.78.16.8630\u20108640.2004"},{"key":"e_1_2_12_14_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0402690101"},{"key":"e_1_2_12_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2007.12.080"},{"key":"e_1_2_12_16_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.85.23.8973"},{"key":"e_1_2_12_17_1","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/17.1.183"},{"key":"e_1_2_12_18_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.200390047"},{"key":"e_1_2_12_19_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.200460028"},{"key":"e_1_2_12_20_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi971470h"},{"key":"e_1_2_12_21_1","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.aaz1590"},{"key":"e_1_2_12_22_1","first-page":"137","volume-title":"Methods in Enzymology","author":"Troilo F.","year":"2018"},{"key":"e_1_2_12_23_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.pep.2006.12.006"},{"key":"e_1_2_12_24_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.2336"},{"key":"e_1_2_12_25_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.354"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70479","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpz1.70479","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70479","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T17:55:12Z","timestamp":1792605312000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70479"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,10]]},"references-count":24,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2026,10]]}},"alternative-id":["10.1002\/cpz1.70479"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70479","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10]]},"article-number":"e70479"}}