{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:28:32Z","timestamp":1785414512422,"version":"3.56.0"},"posted":{"date-parts":[[2025,12,4]]},"group-title":"Biophysics","reference-count":68,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,4]],"date-time":"2025-12-04T00:00:00Z","timestamp":1764806400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["CHE-2528384"],"award-info":[{"award-number":["CHE-2528384"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"award":["MCB-2306190"],"award-info":[{"award-number":["MCB-2306190"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R35GM122603"],"award-info":[{"award-number":["R35GM122603"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R00AG084926"],"award-info":[{"award-number":["R00AG084926"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,12,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  It has been hypothesized that while random sequences are unlikely to fold into proteins of the length of globular proteins, repeated random sequences are more likely to adopt stably folded structures, with implications for molecular evolution. We used structure prediction methods to determine the foldability of approximately 120-residue sequences composed of 5-to 60-residue random repeats. With repeats of less than 30-residues, sequences were frequently discovered (1\u201312%) that fold with high confidence. For less than 60-residue repeats, we frequently observe \u03b2-solenoids, similar to those seen in natural proteins. We observe solenoids stabilized by apolar packing as well as ones stabilized by polar interactions with Ca\n                  <jats:sup>2+<\/jats:sup>\n                  in the core of the structure as in natural RTX domains. Helical bundles were observed with high frequency when insertions or deletions (INDELs) were included between blocks of repeating sequences. We also observed a new super-secondary structure consisting of a tightly wound \u03b1-helical screw, and experimentally confirmed its stability and structure by CD spectroscopy and X-ray crystallography. Thus, structure predictors can discover structures that are well out of the distribution of the data upon which they were trained. Beyond 40-residue repeat lengths very few sequences were predicted to fold. The small number of structures we observed were representative of well-established major classes of tertiary structures; greater sampling would be needed to discover novel structures from a random distribution. These studies illuminate dark matter regions of protein structure space and support previous predictions that proteins evolved through the assortment of shorter peptide sequences.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance statement<\/jats:title>\n                  <jats:p>The availability of powerful and accurate programs for predicting protein three-dimensional structures enables one to ask fundamental questions concerning the origin of folded functional proteins during evolution. We show that 120-residue proteins composed of random sequences repeated in tandem are predicted to be much more likely to fold than fully random proteins. These studies validate previous predictions that proteins evolved through the repetition and assortment of short peptide sequences. Also, some of the predicted structures represent novel conformations, which were confirmed experimentally. These findings advance our understanding of molecular evolution and have implications for design of novel proteins.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2025.12.04.691947","type":"posted-content","created":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T12:41:56Z","timestamp":1765197716000},"source":"Crossref","is-referenced-by-count":2,"title":["The Emergence of Novel Versus Known Three-Dimensional Structures from Random Sequences"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2534-0242","authenticated-orcid":false,"given":"Rose","family":"Yang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2524-2201","authenticated-orcid":false,"given":"Hyunjun","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anton","family":"Davydenko","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-0125-7529","authenticated-orcid":false,"given":"Zack","family":"Mawaldi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5831-1464","authenticated-orcid":false,"given":"Rian","family":"Kormos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5892-0155","authenticated-orcid":false,"given":"Dru","family":"Myerscough","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yibing","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4745-263X","authenticated-orcid":false,"given":"William F.","family":"DeGrado","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025121010550764000_2025.12.04.691947v2.1","unstructured":"Levinthal C (1969) How to fold graciously. In M\u00f6ssbauer Spectroscopy in Biological Systems, Proceedings of a meeting held at Allerton House, Monticello, IL, eds. Debrunner P , Tsibris JCM , M\u00fcnck E . (University of Illinois Press, Urbana), pp. 22\u201324."},{"key":"2025121010550764000_2025.12.04.691947v2.2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.89.1.20"},{"key":"2025121010550764000_2025.12.04.691947v2.3","doi-asserted-by":"publisher","DOI":"10.1126\/science.152.3720.363"},{"key":"2025121010550764000_2025.12.04.691947v2.4","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2016239118"},{"key":"2025121010550764000_2025.12.04.691947v2.5","doi-asserted-by":"publisher","DOI":"10.1002\/bies.10321"},{"key":"2025121010550764000_2025.12.04.691947v2.6","doi-asserted-by":"publisher","DOI":"10.1038\/nrg2808"},{"key":"2025121010550764000_2025.12.04.691947v2.7","doi-asserted-by":"publisher","DOI":"10.7554\/{eLife}.09410"},{"key":"2025121010550764000_2025.12.04.691947v2.8","doi-asserted-by":"publisher","DOI":"10.1126\/science.1169375"},{"key":"2025121010550764000_2025.12.04.691947v2.9","first-page":"1159","article-title":"Diversity and features of proteins with structural repeats","volume":"15","year":"2023","journal-title":"Biophys Rev"},{"key":"2025121010550764000_2025.12.04.691947v2.10","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2315000121"},{"key":"2025121010550764000_2025.12.04.691947v2.11","first-page":"e70054","article-title":"From duplication to fusion: Expanding Dayhoff\u2019s model of protein evolution","volume":"34","year":"2025","journal-title":"Protein Sci"},{"key":"2025121010550764000_2025.12.04.691947v2.12","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biophys.29.1.105"},{"key":"2025121010550764000_2025.12.04.691947v2.13","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2005.06.005"},{"key":"2025121010550764000_2025.12.04.691947v2.14","doi-asserted-by":"publisher","DOI":"10.1016\/S0065-3233(06)73003-0"},{"key":"2025121010550764000_2025.12.04.691947v2.15","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biophys-102220-083020"},{"key":"2025121010550764000_2025.12.04.691947v2.16","first-page":"321","article-title":"Frustration, function, and folding","volume":"50","year":"2021","journal-title":"Annu Rev Biophys"},{"key":"2025121010550764000_2025.12.04.691947v2.17","doi-asserted-by":"publisher","DOI":"10.1126\/science.3043666"},{"key":"2025121010550764000_2025.12.04.691947v2.18","doi-asserted-by":"publisher","DOI":"10.1038\/nature16162"},{"key":"2025121010550764000_2025.12.04.691947v2.19","first-page":"1129","article-title":"Designed armadillo-repeat proteins as modular peptide-binding scaffolds","volume":"112","year":"2015","journal-title":"Proc Natl Acad Sci USA"},{"key":"2025121010550764000_2025.12.04.691947v2.20","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-017-06332-7"},{"key":"2025121010550764000_2025.12.04.691947v2.21","first-page":"e4301","article-title":"Functionalization of a symmetric protein scaffold: Redundant folding nuclei and alternative oligomeric folding pathways","volume":"31","year":"2022","journal-title":"Protein Sci"},{"key":"2025121010550764000_2025.12.04.691947v2.22","doi-asserted-by":"publisher","DOI":"10.1021\/nn5056089"},{"key":"2025121010550764000_2025.12.04.691947v2.23","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1525308113"},{"key":"2025121010550764000_2025.12.04.691947v2.24","unstructured":"Pretorius D , Nikov GI , Washio K , Florent S-W , Taunt HN , Ovchinnikov S , Murray JW (2025) Designing novel solenoid proteins with in silico evolution. bioRxiv: 2025.04.23.646631."},{"key":"2025121010550764000_2025.12.04.691947v2.25","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-04184-w"},{"issue":"27","key":"2025121010550764000_2025.12.04.691947v2.26","first-page":"e2509015122","article-title":"In silico evolution of globular protein folds from random sequences","volume":"122","year":"2025","journal-title":"Proc Natl Acad Sci USA"},{"key":"2025121010550764000_2025.12.04.691947v2.27","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2014.09.007"},{"key":"2025121010550764000_2025.12.04.691947v2.28","doi-asserted-by":"publisher","DOI":"10.1016\/S1359-0278(98)00021-2"},{"key":"2025121010550764000_2025.12.04.691947v2.29","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0509379103"},{"key":"2025121010550764000_2025.12.04.691947v2.30","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.2c12987"},{"key":"2025121010550764000_2025.12.04.691947v2.31","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2012.085"},{"key":"2025121010550764000_2025.12.04.691947v2.32","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-024-01638-w"},{"key":"2025121010550764000_2025.12.04.691947v2.33","first-page":"349","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"631","year":"2024","journal-title":"Nature"},{"key":"2025121010550764000_2025.12.04.691947v2.34","doi-asserted-by":"publisher","DOI":"10.1021\/bi052130w"},{"key":"2025121010550764000_2025.12.04.691947v2.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2011.08.009"},{"key":"2025121010550764000_2025.12.04.691947v2.36","first-page":"638","article-title":"Current understanding of the structure and function of pentapeptide repeat proteins","volume":"11","year":"2021","journal-title":"Biomolecules"},{"key":"2025121010550764000_2025.12.04.691947v2.37","doi-asserted-by":"publisher","DOI":"10.1016\/0969-2126(93)90013-7"},{"key":"2025121010550764000_2025.12.04.691947v2.38","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2006.02.039"},{"key":"2025121010550764000_2025.12.04.691947v2.39","doi-asserted-by":"publisher","DOI":"10.1038\/nature05695"},{"key":"2025121010550764000_2025.12.04.691947v2.40","first-page":"7320","article-title":"Glutamine side chain \u00b9\u00b3C=\u00b9\u2078O as a non-perturbative IR probe of amyloid fibril hydration and assembly","volume":"141","year":"2019","journal-title":"J Am Chem Soc"},{"key":"2025121010550764000_2025.12.04.691947v2.41","doi-asserted-by":"publisher","DOI":"10.1038\/nchem.1894"},{"key":"2025121010550764000_2025.12.04.691947v2.42","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gki524"},{"key":"2025121010550764000_2025.12.04.691947v2.43","first-page":"898","article-title":"Exploring \u201cdark-matter\u201d protein folds using deep learning","volume":"15","year":"2024","journal-title":"Cell Syst"},{"key":"2025121010550764000_2025.12.04.691947v2.44","doi-asserted-by":"publisher","DOI":"10.1038\/nature11600"},{"key":"2025121010550764000_2025.12.04.691947v2.45","doi-asserted-by":"publisher","DOI":"10.1126\/science.3381086"},{"key":"2025121010550764000_2025.12.04.691947v2.46","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-06728-8"},{"key":"2025121010550764000_2025.12.04.691947v2.47","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae1087"},{"key":"2025121010550764000_2025.12.04.691947v2.48","doi-asserted-by":"publisher","DOI":"10.1021\/nl035159z"},{"key":"2025121010550764000_2025.12.04.691947v2.49","doi-asserted-by":"publisher","DOI":"10.1096\/fj.04-3256hyp"},{"key":"2025121010550764000_2025.12.04.691947v2.50","first-page":"1499","article-title":"Amyloid and the origin of life: self-replicating catalytic amyloids as prebiotic informational and protometabolic entities","volume":"75","year":"2018","journal-title":"Cell Mol Life Sci"},{"key":"2025121010550764000_2025.12.04.691947v2.51","doi-asserted-by":"publisher","DOI":"10.1038\/nchem.861"},{"key":"2025121010550764000_2025.12.04.691947v2.52","doi-asserted-by":"publisher","DOI":"10.1039\/C8CS00080H"},{"key":"2025121010550764000_2025.12.04.691947v2.53","first-page":"907","article-title":"Catalytic amyloids","volume":"4","year":"2022","journal-title":"Trends Chem"},{"issue":"4","key":"2025121010550764000_2025.12.04.691947v2.54","first-page":"197","article-title":"A designed, phase changing RTX-based peptide for efficient bioseparations","volume":"54","year":"2013","journal-title":"BioTechniques"},{"key":"2025121010550764000_2025.12.04.691947v2.55","doi-asserted-by":"publisher","DOI":"10.1038\/nchem.2122"},{"key":"2025121010550764000_2025.12.04.691947v2.56","doi-asserted-by":"publisher","DOI":"10.1021\/nn5056089"},{"key":"2025121010550764000_2025.12.04.691947v2.57","doi-asserted-by":"publisher","DOI":"10.1039\/c4ob00447g"},{"key":"2025121010550764000_2025.12.04.691947v2.58","doi-asserted-by":"publisher","DOI":"10.1016\/j.csbj.2025.01.016"},{"key":"2025121010550764000_2025.12.04.691947v2.59","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-6976.2010.00231.x"},{"key":"2025121010550764000_2025.12.04.691947v2.60","first-page":"107381","article-title":"Ancestral sequence reconstruction of ancient lipase from family I.3 bacterial lipolytic enzymes","volume":"168","year":"2022","journal-title":"Mol Phylogenet Evol"},{"key":"2025121010550764000_2025.12.04.691947v2.61","first-page":"e0143948","article-title":"Towards prebiotic catalytic amyloids using high-throughput screening","volume":"10","year":"2015","journal-title":"PLoS One"},{"key":"2025121010550764000_2025.12.04.691947v2.62","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2010.08.058"},{"key":"2025121010550764000_2025.12.04.691947v2.63","doi-asserted-by":"publisher","DOI":"10.1110\/ps.9.9.1753"},{"key":"2025121010550764000_2025.12.04.691947v2.64","doi-asserted-by":"publisher","DOI":"10.1126\/science.ade2574"},{"key":"2025121010550764000_2025.12.04.691947v2.65","doi-asserted-by":"publisher","DOI":"10.1107\/S0021889807021206"},{"key":"2025121010550764000_2025.12.04.691947v2.66","doi-asserted-by":"publisher","DOI":"10.1107\/S090744490705024X"},{"key":"2025121010550764000_2025.12.04.691947v2.67","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444910007493"},{"key":"2025121010550764000_2025.12.04.691947v2.68","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444912001308"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2025.12.04.691947","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T18:55:11Z","timestamp":1765392911000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2025.12.04.691947"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,4]]},"references-count":68,"URL":"https:\/\/doi.org\/10.64898\/2025.12.04.691947","relation":{},"subject":[],"published":{"date-parts":[[2025,12,4]]},"subtype":"preprint"}}