{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T12:09:19Z","timestamp":1778328559387,"version":"3.51.4"},"reference-count":87,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T00:00:00Z","timestamp":1774224000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T00:00:00Z","timestamp":1774224000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2026,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    We present the structural and functional characterization of a single\u2010stranded DNA\u2010binding protein (SSB\u2010M5) identified from a hot spring metagenome in Vatnaj\u00f6kull National Park, Iceland. This small protein (136 aa; 15,695\u2009Da) shares 100% amino acid sequence identity with two previously uncharacterized SSBs from hyperthermophilic\n                    <jats:italic>Fervidobacterium<\/jats:italic>\n                    species. Functional complementation assay demonstrated that SSB\u2010M5 can substitute for\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    SSB in an\n                    <jats:italic>ssb<\/jats:italic>\n                    <jats:sup>\u2212<\/jats:sup>\n                    mutant strain, confirming its biological activity. A recombinant C\u2010terminally His\u2010tagged SSB\u2010M5 was overproduced, purified to homogeneity, and subjected to structural, biochemical, and biophysical analysis. The crystal structure revealed that SSB\u2010M5 forms a dimer through a crystallographic twofold axis, with each monomer contributing to a large antiparallel\n                    <jats:italic>\u03b2<\/jats:italic>\n                    \u2010sheet. The flat surfaces of the\n                    <jats:italic>\u03b2<\/jats:italic>\n                    \u2010sheets from the two dimers are packed together via a second crystallographic twofold axis, forming a tetramer that serves as the functional unit of the SSB\u2010M5. Electrophoretic mobility shift assays showed that SSB\u2010M5, after heat treatment up to 100\u00b0C, forms stable DNA\u2010protein complexes with the (dT)\n                    <jats:sub>40<\/jats:sub>\n                    oligo. Quantitative analyses revealed that SSB\u2010M5 binds (dT)\n                    <jats:sub>70<\/jats:sub>\n                    oligonucleotide with very high affinity (\n                    <jats:italic>K<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>D<\/jats:italic>\n                    <\/jats:sub>\n                    \u2009=\u200972\u2009\u00b1\u20096 pM). Hill analysis indicated cooperative binding, yielding an EC\n                    <jats:sub>50<\/jats:sub>\n                    of 141 pM and a Hill coefficient of 2. Moreover, inclusion of SSB\u2010M5 in PCR reactions significantly enhanced amplification by eliminating non\u2010specific products. Together, these findings identify SSB\u2010M5 as a hyperthermostable, high\u2010affinity single\u2010stranded DNA\u2010binding protein with potential applications in molecular biology and biotechnology.\n                  <\/jats:p>","DOI":"10.1002\/pro.70538","type":"journal-article","created":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T13:44:46Z","timestamp":1774273486000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Structural and functional characterization of a hyperthermostable single\u2010stranded\n                    <scp>DNA<\/scp>\n                    \u2010binding protein from a hot spring metagenome"],"prefix":"10.1002","volume":"35","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0194-0673","authenticated-orcid":false,"given":"Olesia","family":"Werbowy","sequence":"first","affiliation":[{"name":"Department of Microbiology Faculty of Biology, University of Gdansk  Gdansk Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7264-6316","authenticated-orcid":false,"given":"Maria","family":"H\u00e5kansson","sequence":"additional","affiliation":[{"name":"SARomics Biostructures  Lund Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sebastian","family":"Dorawa","sequence":"additional","affiliation":[{"name":"Laboratory of Extremophiles Biology, Department of Microbiology Faculty of Biology, University of Gdansk  Gdansk Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aleksandra","family":"Stefa\u0144ska\u2010Ka\u017amierczak","sequence":"additional","affiliation":[{"name":"A&amp;A Biotechnology  Gdansk Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"L. Anders","family":"Svensson","sequence":"additional","affiliation":[{"name":"SARomics Biostructures  Lund Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Salam","family":"Al\u2010Karadaghi","sequence":"additional","affiliation":[{"name":"SARomics Biostructures  Lund Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Agata","family":"Jurczak\u2010Kurek","sequence":"additional","affiliation":[{"name":"Laboratory of Molecular Evolution and Bioinformatics, Department of Evolutionary Genetics and Biosystematics University of Gdansk  Gdansk Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karolina","family":"Kwiatkowska\u2010Semrau","sequence":"additional","affiliation":[{"name":"Laboratory of Structural Biology Institute of Biotechnology, Intercollegiate Faculty of Biotechnology UG&amp;MUG  Gdansk"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2871-8060","authenticated-orcid":false,"given":"Magdalena","family":"Plotka","sequence":"additional","affiliation":[{"name":"Department of Microbiology Faculty of Biology, University of Gdansk  Gdansk Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Olafur H.","family":"Fridjonsson","sequence":"additional","affiliation":[{"name":"Matis ohf  Reykjavik Iceland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gudmundur O.","family":"Hreggvidsson","sequence":"additional","affiliation":[{"name":"Matis ohf  Reykjavik Iceland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Arnth\u00f3r","family":"Aevarsson","sequence":"additional","affiliation":[{"name":"Matis ohf  Reykjavik Iceland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"S\u0142awomir","family":"D\u0105browski","sequence":"additional","affiliation":[{"name":"A&amp;A Biotechnology  Gdansk Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6598-8449","authenticated-orcid":false,"given":"Anna\u2010Karina","family":"Kaczorowska","sequence":"additional","affiliation":[{"name":"Collection of Plasmids and Microorganisms, Department of Microbiology, Faculty of Biology, University of Gdansk  Gdansk Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2305-2677","authenticated-orcid":false,"given":"Tadeusz","family":"Kaczorowski","sequence":"additional","affiliation":[{"name":"Laboratory of Extremophiles Biology, Department of Microbiology Faculty of Biology, University of Gdansk  Gdansk Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2026,3,23]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1093\/femsle\/fnab067"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/25.17.3389"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1099\/00207713-46-1-265"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja042394q"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.pbiomolbio.2016.11.001"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/385176a0"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/jmr.657"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(76)90527-3"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mie.2015.05.001"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(19)70317-2"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2009.03.022"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(17)43529-0"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1006\/prep.1999.1044"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1099\/00221287-148-10-3307"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/s1046-5928(02)00504-1"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2025.108230"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms252413544"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms23147945"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444910007493"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444913000061"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkl550"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00203-006-0128-2"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.pep.2006.11.006"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00792-006-0011-8"},{"key":"e_1_2_8_26_1","doi-asserted-by":"publisher","DOI":"10.18388\/abp.2007_3272"},{"key":"e_1_2_8_27_1","doi-asserted-by":"publisher","DOI":"10.1099\/mic.0.025023-0"},{"key":"e_1_2_8_28_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gku259"},{"key":"e_1_2_8_29_1","first-page":"iv","article-title":"The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves","volume":"40","author":"Hill AV","year":"1910","journal-title":"J Physiol"},{"key":"e_1_2_8_30_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(17)34538-6"},{"key":"e_1_2_8_31_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.4585"},{"key":"e_1_2_8_32_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molstruc.2014.03.009"},{"key":"e_1_2_8_33_1","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(84)90539-7"},{"key":"e_1_2_8_34_1","doi-asserted-by":"publisher","DOI":"10.1016\/s0968-0004(01)01918-1"},{"key":"e_1_2_8_35_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444907002661"},{"key":"e_1_2_8_36_1","doi-asserted-by":"publisher","DOI":"10.1128\/mr.58.3.401-465.1994"},{"key":"e_1_2_8_37_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkab295"},{"key":"e_1_2_8_38_1","doi-asserted-by":"publisher","DOI":"10.1093\/protein\/13.3.179"},{"key":"e_1_2_8_39_1","doi-asserted-by":"publisher","DOI":"10.18388\/abp.2005_3416"},{"key":"e_1_2_8_40_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2006.02.070"},{"key":"e_1_2_8_41_1","doi-asserted-by":"publisher","DOI":"10.1515\/BC.2002.149"},{"key":"e_1_2_8_42_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bi.63.070194.002523"},{"key":"e_1_2_8_43_1","doi-asserted-by":"publisher","DOI":"10.1016\/0022-2836(81)90337-5"},{"key":"e_1_2_8_44_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0711919105"},{"key":"e_1_2_8_45_1","doi-asserted-by":"publisher","DOI":"10.1093\/oxfordjournals.jbchem.a022611"},{"key":"e_1_2_8_46_1","doi-asserted-by":"publisher","DOI":"10.1128\/mr.54.4.342-380.1990"},{"key":"e_1_2_8_47_1","doi-asserted-by":"publisher","DOI":"10.1016\/s0022-2836(75)80012-x"},{"key":"e_1_2_8_48_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1097-2765(03)00188-6"},{"key":"e_1_2_8_49_1","doi-asserted-by":"publisher","DOI":"10.1016\/0005-2744(69)90420-3"},{"key":"e_1_2_8_50_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444911001314"},{"key":"e_1_2_8_51_1","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1993.tb05726.x"},{"key":"e_1_2_8_52_1","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2180-14-91"},{"key":"e_1_2_8_53_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0126563"},{"key":"e_1_2_8_54_1","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2180-10-260"},{"key":"e_1_2_8_55_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.micres.2013.07.010"},{"key":"e_1_2_8_56_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mcp.2004.11.006"},{"key":"e_1_2_8_57_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkg865"},{"key":"e_1_2_8_58_1","doi-asserted-by":"publisher","DOI":"10.3390\/v11070657"},{"key":"e_1_2_8_59_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.173.8.2720-2723.1991"},{"key":"e_1_2_8_60_1","doi-asserted-by":"publisher","DOI":"10.1093\/emboj\/19.23.6536"},{"key":"e_1_2_8_61_1","doi-asserted-by":"publisher","DOI":"10.1038\/77943"},{"key":"e_1_2_8_62_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF02745882"},{"key":"e_1_2_8_63_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2007.03.079"},{"key":"e_1_2_8_64_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi9705321"},{"key":"e_1_2_8_65_1","volume-title":"Molecular cloning: a laboratory manual","author":"Sambrook J","year":"1989"},{"key":"e_1_2_8_66_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.78.7.4274"},{"key":"e_1_2_8_67_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-3495(00)76713-0"},{"key":"e_1_2_8_68_1","doi-asserted-by":"publisher","DOI":"10.1038\/376362a0"},{"key":"e_1_2_8_69_1","doi-asserted-by":"publisher","DOI":"10.1080\/10409230802341296"},{"key":"e_1_2_8_70_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt1238"},{"key":"e_1_2_8_71_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.69.12.3537"},{"key":"e_1_2_8_72_1","doi-asserted-by":"publisher","DOI":"10.1007\/s13353-015-0314-5"},{"key":"e_1_2_8_73_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiotec.2014.04.015"},{"key":"e_1_2_8_74_1","doi-asserted-by":"publisher","DOI":"10.2144\/00286ir01"},{"key":"e_1_2_8_75_1","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1097-0282(1997)44:4<405::AID-BIP5>3.0.CO;2-L"},{"key":"e_1_2_8_76_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.08193"},{"key":"e_1_2_8_77_1","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/msab120"},{"key":"e_1_2_8_78_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/22.22.4673"},{"key":"e_1_2_8_79_1","doi-asserted-by":"publisher","DOI":"10.1016\/s1359-0278(97)00062-x"},{"key":"e_1_2_8_80_1","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.1997.1042"},{"key":"e_1_2_8_81_1","doi-asserted-by":"publisher","DOI":"10.1128\/jb.151.1.186-192.1982"},{"key":"e_1_2_8_82_1","doi-asserted-by":"publisher","DOI":"10.1186\/1472-6750-8-2"},{"key":"e_1_2_8_83_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.plasmid.2014.12.002"},{"key":"e_1_2_8_84_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms1093"},{"key":"e_1_2_8_85_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(20)71283-4"},{"key":"e_1_2_8_86_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444913015308"},{"key":"e_1_2_8_87_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gki310"},{"key":"e_1_2_8_88_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.66.1.61"}],"container-title":["Protein Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/pro.70538","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/pro.70538","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/pro.70538","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T11:20:40Z","timestamp":1778325640000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/pro.70538"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,23]]},"references-count":87,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2026,4]]}},"alternative-id":["10.1002\/pro.70538"],"URL":"https:\/\/doi.org\/10.1002\/pro.70538","archive":["Portico"],"relation":{},"ISSN":["0961-8368","1469-896X"],"issn-type":[{"value":"0961-8368","type":"print"},{"value":"1469-896X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,23]]},"assertion":[{"value":"2025-11-04","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-03-05","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-03-23","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e70538"}}