{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T21:19:25Z","timestamp":1792790365755,"version":"4.5.0"},"posted":{"date-parts":[[2026,9,20]]},"group-title":"Biophysics","reference-count":25,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,20]],"date-time":"2026-09-20T00:00:00Z","timestamp":1789862400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"ational Institutes of Health, National Institute of General Medical Sciences","award":["R35GM161360"],"award-info":[{"award-number":["R35GM161360"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,20]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Selective molecular recognition of dynamic nucleic acid structures is fundamental to DNA replication, yet the physical mechanisms by which intrinsically disordered protein domains achieve this selectivity remain poorly understood. Here, we investigate how the intrinsically disordered C-terminal domain (CTD) of the bacteriophage T4 single-stranded DNA-binding protein gp32 contributes to the recognition of ss\u2013dsDNA replication fork junctions. Using absorbance, circular dichroism, and two-dimensional fluorescence spectroscopy (2DFS) of model DNA replication junctions containing an exciton-coupled (Cy3)\n                  <jats:sub>2<\/jats:sub>\n                  probe, we quantified their average solution structures and conformational heterogeneity. Protein-free DNA junctions exhibit distinct conformational distributions that depend on ssDNA arm lengths and local base sequence, demonstrating that this heterogeneity exists prior to protein binding. Although both wild-type gp32 and the CTD-truncated mutant gp32 I* substantially remodel the average junction structure, only wild-type gp32 preserves and amplifies these differences in conformational heterogeneity. These results support a model in which the intrinsically disordered CTD promotes selective molecular recognition by redistributing populations among pre-existing DNA conformational states rather than by stabilizing a single protein-bound conformation. More broadly, our findings suggest that intrinsically disordered protein domains can recognize dynamic nucleic acid substrates by amplifying conformational heterogeneity already present within their intrinsic conformational landscapes.\n                <\/jats:p>","DOI":"10.64898\/2026.09.18.752737","type":"posted-content","created":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T04:55:12Z","timestamp":1789966512000},"source":"Crossref","is-referenced-by-count":0,"title":["The intrinsically disordered C-terminal domain of bacteriophage T4 gp32 amplifies pre-existing conformational heterogeneity at DNA replication junctions"],"prefix":"10.64898","author":[{"given":"Lulu","family":"Enkhbaatar","sequence":"first","affiliation":[{"name":"Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA"},{"name":"Center for Optical, Molecular and Quantum Science, University of Oregon, Eugene, OR 97403, USA"},{"name":"Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR 97403, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter H.","family":"von Hippel","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA"},{"name":"Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR 97403, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2109-327X","authenticated-orcid":false,"given":"Andrew H.","family":"Marcus","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA"},{"name":"Center for Optical, Molecular and Quantum Science, University of Oregon, Eugene, OR 97403, USA"},{"name":"Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR 97403, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026092317001030000_2026.09.18.752737v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/nature01407"},{"key":"2026092317001030000_2026.09.18.752737v1.2","doi-asserted-by":"crossref","first-page":"10730","DOI":"10.1021\/acs.jpcb.3c06463","article-title":"Studies of DNA \u2018breathing\u2019 by polarization-sweep single-molecule fluorescence microscopy of exciton-coupled (iCy3)2 dimer-labeled DNA fork constructs","volume":"127","year":"2023","journal-title":"J. Phys. Chem. B"},{"key":"2026092317001030000_2026.09.18.752737v1.3","doi-asserted-by":"crossref","first-page":"gkaf047","DOI":"10.1093\/nar\/gkaf047","article-title":"Site-specific free energy surface parameters from single-molecule fluorescence measurements of exciton-coupled (iCy3)2 dimer probes positioned at DNA replication fork junctions","volume":"53","year":"2025","journal-title":"Nucleic Acids Res"},{"key":"2026092317001030000_2026.09.18.752737v1.4","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1186\/1743-422X-7-359","article-title":"Structural analysis of bacteriophage T4 DNA replication: A review in the Virology Journal series on bacteriophage T4 and its relatives","volume":"7","year":"2010","journal-title":"Virology J"},{"key":"2026092317001030000_2026.09.18.752737v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2024.168544"},{"key":"2026092317001030000_2026.09.18.752737v1.6","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1021\/acs.biochem.5c00541","article-title":"Functional integration of the bacteriophage T4 DNA replication complex: The multiple roles of the ssDNA binding protein (gp32)","volume":"65","year":"2026","journal-title":"Biochemistry"},{"key":"2026092317001030000_2026.09.18.752737v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/376362a0"},{"key":"2026092317001030000_2026.09.18.752737v1.8","doi-asserted-by":"crossref","first-page":"2463","DOI":"10.1016\/S0021-9258(17)38096-1","article-title":"Structural changes in the T4 gene 32 protein induced by DNA polynucleotides","volume":"253","year":"1978","journal-title":"J. Biol. Chem"},{"key":"2026092317001030000_2026.09.18.752737v1.9","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.89.3.1050"},{"key":"2026092317001030000_2026.09.18.752737v1.10","doi-asserted-by":"crossref","first-page":"11484","DOI":"10.1016\/S0021-9258(19)70317-2","article-title":"Proteolytic removal of the COOH terminus of the T4 gene 32 helix-destabilizing protein alters the T4 in vitro replication complex","volume":"255","year":"1980","journal-title":"J. Biol. Chem"},{"key":"2026092317001030000_2026.09.18.752737v1.11","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1038\/s41580-023-00673-0","article-title":"The molecular basis for cellular function of intrinsically disordered protein regions","volume":"25","year":"2024","journal-title":"Nature Reviews"},{"key":"2026092317001030000_2026.09.18.752737v1.12","doi-asserted-by":"publisher","DOI":"10.1038\/s41576-025-00816-3"},{"key":"2026092317001030000_2026.09.18.752737v1.13","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0194357"},{"key":"2026092317001030000_2026.09.18.752737v1.14","doi-asserted-by":"crossref","first-page":"085101","DOI":"10.1063\/1.5020084","article-title":"Temperature-dependent conformations of exciton-coupled Cy3 dimers in double-stranded DNA","volume":"148","year":"2018","journal-title":"J. Chem. Phys"},{"key":"2026092317001030000_2026.09.18.752737v1.15","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad1163"},{"key":"2026092317001030000_2026.09.18.752737v1.16","doi-asserted-by":"publisher","DOI":"10.1039\/C8FD00245B"},{"key":"2026092317001030000_2026.09.18.752737v1.17","doi-asserted-by":"crossref","first-page":"045101","DOI":"10.1063\/5.0076261","article-title":"Temperature-dependent local conformations and conformational distributions of cyanine dimer labeled single-stranded\u2013double-stranded DNA junctions by 2D fluorescence spectroscopy","volume":"156","year":"2022","journal-title":"J. Chem. Phys"},{"key":"2026092317001030000_2026.09.18.752737v1.18","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkw863"},{"key":"2026092317001030000_2026.09.18.752737v1.19","first-page":"7215","article-title":"DNA \u201cmelting\u201d proteins","volume":"251","year":"1976","journal-title":"J. Biol. Chem"},{"key":"2026092317001030000_2026.09.18.752737v1.20","doi-asserted-by":"publisher","DOI":"10.1063\/1.2800560"},{"key":"2026092317001030000_2026.09.18.752737v1.21","doi-asserted-by":"crossref","first-page":"7852","DOI":"10.1021\/acs.jpca.1c05556","article-title":"Accurate modeling of excitonic coupling in cyanine dye Cy3","volume":"125","year":"2021","journal-title":"J. Phys. Chem. A"},{"key":"2026092317001030000_2026.09.18.752737v1.22","doi-asserted-by":"publisher","DOI":"10.1002\/bip.22347"},{"key":"2026092317001030000_2026.09.18.752737v1.23","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2012.02.028"},{"key":"2026092317001030000_2026.09.18.752737v1.24","unstructured":"Enkhbaatar, L. , D. Heussman , P. H. von Hippel , and A. H. Marcus . (2026) Quantitative lineshape analysis for 2DFS and absorbance\/CD spectra. Zenodo, 10.5281\/zenodo.22649211."},{"key":"2026092317001030000_2026.09.18.752737v1.25","unstructured":"Heussman, D. , A. Marcus , and P. H. von Hippel . (2026) 2DFS Response function library. Zenodo, 10.5281\/zenodo.22102458."}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.09.18.752737","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T00:00:18Z","timestamp":1790208018000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.09.18.752737"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,20]]},"references-count":25,"URL":"https:\/\/doi.org\/10.64898\/2026.09.18.752737","relation":{},"subject":[],"published":{"date-parts":[[2026,9,20]]},"subtype":"preprint"}}