{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T16:23:31Z","timestamp":1787243011211,"version":"build-2736575974"},"reference-count":44,"publisher":"AIP Publishing","issue":"4","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":50,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R35-GM141818"],"award-info":[{"award-number":["R35-GM141818"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["1S10OD030482"],"award-info":[{"award-number":["1S10OD030482"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000161","name":"National Institute of Standards and Technology","doi-asserted-by":"publisher","award":["60NANB22D167"],"award-info":[{"award-number":["60NANB22D167"]}],"id":[{"id":"10.13039\/100000161","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["1S10OD32168-01A1"],"award-info":[{"award-number":["1S10OD32168-01A1"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["pubs.aip.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2026,7,1]]},"abstract":"<jats:p>The SARS-CoV-2 virus causes COVID-19, and several of its gene products have been successfully targeted for antiviral drug development, including the 3C-like protease (3CLpro). The substrate-binding site of 3CLpro exhibits significant conformational plasticity. While available X-ray crystal structures reveal substantial loop variability and molecular dynamics simulations indicate that conformational heterogeneity persists in ligand-bound complexes, the slow-timescale thermodynamic and kinetic landscape of these complexes in solution remains incompletely defined. Using 19F NMR spectroscopy, we characterize a slow-exchange conformational equilibrium in both the covalent nirmatrelvir (NMV) and noncovalent ensitrelvir (ENS) complexes of 3CLpro. For the wild-type 3CLpro\u2013NMV complex at 298\u2009K, joint dual-field line shape and exchange spectroscopy analysis resolves a millisecond-timescale exchange between two distinct states (population ratio\u2009\u223c\u200965:35; kex\u2009\u2248\u200952\u2009s\u22121; \u0394G\u2021\u2009\u2248\u200915\u2009kcal\u2009mol\u22121). Co-existing bound states also persist in both the noncovalent NMV\u2013[C145A] mutant (minor state population, pB\u2009\u2248\u200918%) and wild-type ENS (pB\u2009\u2248\u200938%) complexes, demonstrating that this slow-exchange heterogeneity is an intrinsic property of the ligated protease rather than a consequence of covalent attachment. These dynamics are not readily explained by available crystal structures or conventional microsecond molecular dynamics simulations, suggesting that in solution the inhibited enzyme samples alternative, energetically accessible conformations that are not fully represented in the crystal lattice. This study highlights the utility of solution-state 19F NMR for quantifying low-energy conformational states relevant to drug\u2013target energetics and inhibitor design.<\/jats:p>","DOI":"10.1063\/4.0001210","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T15:25:28Z","timestamp":1787239528000},"update-policy":"https:\/\/doi.org\/10.1063\/aip-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Slowly exchanging bound states of SARS-CoV-2 3CLpro-inhibitor complexes revealed by 19F NMR"],"prefix":"10.1063","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9549-2711","authenticated-orcid":false,"given":"Anna","family":"De Falco","sequence":"first","affiliation":[{"name":"Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2407-9617","authenticated-orcid":false,"given":"Ben","family":"Shurina","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5967-1402","authenticated-orcid":false,"given":"Rebecca","family":"Greene-Cramer","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0335-1573","authenticated-orcid":false,"given":"Theresa A.","family":"Ramelot","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9440-3059","authenticated-orcid":false,"given":"Gaetano T.","family":"Montelione","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"317","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"2026082011244123400_c1","doi-asserted-by":"publisher","first-page":"265","DOI":"10.1038\/s41586-020-2008-3","article-title":"A new coronavirus associated with human respiratory disease in China","volume":"579","year":"2020","journal-title":"Nature"},{"key":"2026082011244123400_c2","doi-asserted-by":"publisher","first-page":"270","DOI":"10.1038\/s41586-020-2012-7","article-title":"A pneumonia outbreak associated with a new coronavirus of probable bat origin","volume":"579","year":"2020","journal-title":"Nature"},{"key":"2026082011244123400_c3","doi-asserted-by":"publisher","first-page":"727","DOI":"10.1056\/NEJMoa2001017","article-title":"A novel coronavirus from patients with pneumonia in China, 2019","volume":"382","year":"2020","journal-title":"N. 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