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We find that the previously identified open-interface trimer conformation is sampled in all eVLP-displayed spike variants studied including sequences from engineered vaccine constructs and native viral sequences. The D614G mutation, which arose early in the pandemic, favors the canonical \u2018closed-interface\u2019 prefusion conformation, potentially mitigating premature S1 shedding in the presence of a cleaved furin site and providing an evolutionary advantage to the virus. Remarkably, furin cleavage at the S1\/S2 boundary allosterically increases the flexibility of the S2\u2019 site, which may facilitate increased TMPRSS2 processing, enhancing viral infectivity. The use of eVLPs in HDX-MS studies provides a powerful platform for studying viral and membrane proteins in near-native environments.<\/jats:p>","DOI":"10.1101\/2025.01.27.635166","type":"posted-content","created":{"date-parts":[[2025,1,28]],"date-time":"2025-01-28T21:30:17Z","timestamp":1738099817000},"source":"Crossref","is-referenced-by-count":3,"title":["The Interplay of Furin Cleavage and D614G in Modulating SARS-CoV-2 Spike Protein Dynamics"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5278-2361","authenticated-orcid":false,"given":"Sophie R.","family":"Shoemaker","sequence":"first","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-3327-7948","authenticated-orcid":false,"given":"Megan","family":"Luo","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1416-4757","authenticated-orcid":false,"given":"Kim-Marie A.","family":"Dam","sequence":"additional","affiliation":[{"name":"Gladstone Institute of Virology, San Francisco, CA, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2998-9735","authenticated-orcid":false,"given":"John E.","family":"Pak","sequence":"additional","affiliation":[{"name":"Chan Zuckerberg Biohub - San Francisco, San Francisco, CA, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4923-9568","authenticated-orcid":false,"given":"Magnus A. 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