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Here, we report on the conformational changes in PR flaps for drug\u2010resistance mutations using\n                    <jats:sup>19<\/jats:sup>\n                    F nuclear magnetic resonance (NMR) spectroscopy. We prepared wild\u2010type (wt) PR and its three mutant variants containing 4, 10, or 11 mutations, namely 4Mut, 10Mut, and 11Mut, that were previously found in a viral passage study against darunavir (DRV). The proteins were uniformly labeled with\n                    <jats:sup>15<\/jats:sup>\n                    N and with\n                    <jats:sup>19<\/jats:sup>\n                    F amino\u2010acid specifically in the 5\u2010indole positions of two conserved tryptophan residues, W6 and W42, which are located near the dimer interface and at the edge of the flap region of PR, respectively. In solution,\n                    <jats:sup>19<\/jats:sup>\n                    F NMR spectra of these proteins in the inhibitor\u2010free forms showed a downfield shift of the W42 resonance as the number of mutations increased. In the DRV\u2010bound form, the W42 resonance again shifted with increasing number of mutations and, importantly, split into two peaks, reflecting different local electronic environments of the two flaps of the dimer. Solid\u2010state\n                    <jats:sup>19<\/jats:sup>\n                    F magic angle spinning (MAS) NMR spectra of wt\u2010PR and 10Mut also exhibited two W42 resonances in DRV\u2010bound crystalline forms. These observations indicate that drug\u2010resistance mutations increase the asymmetry between the two flaps in the PR\u2010inhibitor interaction in the inhibitor\u2010bound forms.\n                  <\/jats:p>","DOI":"10.1002\/pro.70736","type":"journal-article","created":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T11:18:18Z","timestamp":1785928698000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Conformational asymmetry of human immunodeficiency virus\u20101 protease flaps increases along the darunavir drug resistance pathway"],"prefix":"10.1002","volume":"35","author":[{"given":"Michel","family":"Guerrero","sequence":"first","affiliation":[{"name":"Department of Structural Biology University of Pittsburgh School of Medicine  Pittsburgh Pennsylvania 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