{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T17:09:57Z","timestamp":1790615397802,"version":"4.1.0"},"reference-count":73,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T00:00:00Z","timestamp":1766534400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001411","name":"Indian Council of Medical Research","doi-asserted-by":"crossref","award":["12(67)\/2022"],"award-info":[{"award-number":["12(67)\/2022"]}],"id":[{"id":"10.13039\/501100001411","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Builder Grant","award":["BT\/INF\/22\/SP45072\/2022"],"award-info":[{"award-number":["BT\/INF\/22\/SP45072\/2022"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>The RNA Recognition Motif (RRM) domain of the Ewing sarcoma (EWS) protein plays a pivotal role in RNA binding and gene regulation, being crucial for its function. However, its structural dynamics are yet to be revealed. Herein, we performed 5.5 \u03bcs cumulative molecular dynamics (MD) simulations to investigate the unfolding pathways of the EWS-RRM domain in urea and DMSO across 300\u2013500 K. The unfolding process was characterized by using free-energy landscape (FEL) analysis, hydrogen-bond occupancy, and Gaussian Mixture Model (GMM) clustering. At lower temperatures (300\u2013350 K), the RRM largely retained its native conformation, while extensive unfolding occurred between 400 and 450 K. Results revealed multiple conformational ensembles: native (N), native-like intermediate (IN), intermediate (I), and unfolded (U) states, underlying the unfolding pathway of RRM. In urea at 400 K, a long-lived I-state dominated, with transient N and IN-populations, whereas in DMSO, the IN-state appeared more stable, that transitioned into tightly packed I-states, reflecting a stepwise unfolding via compact intermediates. At 450 K, the protein reached the U-state in both solvents, though unfolding occurred more readily in urea. This study highlights the solvent-dependent unfolding mechanisms and heterogeneous I-states of EWS-RRM, providing insight into its stability, misfolding, and potential relevance to Ewing sarcoma pathogenesis.<\/jats:p>","DOI":"10.3390\/biom16010033","type":"journal-article","created":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T16:41:19Z","timestamp":1766594479000},"page":"33","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Heterogeneous Folding Intermediates Govern the Conformational Pathway of the RNA Recognition Motif Domain of the Ewing Sarcoma Protein"],"prefix":"10.3390","volume":"16","author":[{"given":"Priyanka","family":"Kataria","sequence":"first","affiliation":[{"name":"Data Science Laboratory, Amity Institute of Integrative Sciences and Health, Amity University Haryana, Gurugram 122413, Haryana, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vishakha","family":"Chaudhary","sequence":"additional","affiliation":[{"name":"Data Science Laboratory, Amity Institute of Integrative Sciences and Health, Amity University Haryana, Gurugram 122413, Haryana, India"},{"name":"Amity Institute of Biotechnology, Amity University Haryana, Gurugram 122413, Haryana, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chandra Bhushan","family":"Mishra","sequence":"additional","affiliation":[{"name":"Department of Pharmacology & Chemical Biology, Baylor College of Medicine, Houston, TX 77030, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3621-5025","authenticated-orcid":false,"given":"Vijay","family":"Kumar","sequence":"additional","affiliation":[{"name":"Amity Institute of Biotechnology, Amity University Noida, Noida 201313, Uttar Pradesh, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ravi Datta","family":"Sharma","sequence":"additional","affiliation":[{"name":"School of Biotechnology, Shri Mata Vaishno Devi University, Katra 182320, Jammu and Kashmir, India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4821-1654","authenticated-orcid":false,"given":"Amresh","family":"Prakash","sequence":"additional","affiliation":[{"name":"Data Science Laboratory, Amity Institute of Integrative Sciences and Health, Amity University Haryana, Gurugram 122413, Haryana, India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1938","DOI":"10.1016\/j.bbadis.2018.10.042","article-title":"EWSR1, a multifunctional protein, regulates cellular function and aging via genetic and epigenetic pathways","volume":"1865","author":"Lee","year":"2019","journal-title":"Biochim. 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