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Darunavir (DRV) is a highly potent protease inhibitor (PI) that is oftentimes effective when drug resistance has emerged against first-generation inhibitors. Resistance to darunavir does evolve and requires 10\u201320 amino acid substitutions. The conformational landscapes of six highly characterized HIV-1 protease (PR) constructs that harbor up to 19 DRV-associated mutations were characterized by distance measurements with pulsed electron double resonance (PELDOR) paramagnetic resonance spectroscopy, namely double electron\u2013electron resonance (DEER). The results show that the accumulated substitutions alter the conformational landscape compared to PI-na\u00efve protease where the semi-open conformation is destabilized as the dominant population with open-like states becoming prevalent in many cases. A linear correlation is found between values of the DRV inhibition parameter Ki and the open-like to closed-state population ratio determined from DEER. The nearly 50% decrease in occupancy of the semi-open conformation is associated with reduced enzymatic activity, characterized previously in the literature.<\/jats:p>","DOI":"10.3390\/v12111275","type":"journal-article","created":{"date-parts":[[2020,11,8]],"date-time":"2020-11-08T19:03:37Z","timestamp":1604862217000},"page":"1275","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Darunavir-Resistant HIV-1 Protease Constructs Uphold a Conformational Selection Hypothesis for Drug Resistance"],"prefix":"10.3390","volume":"12","author":[{"given":"Zhanglong","family":"Liu","sequence":"first","affiliation":[{"name":"Department of Chemistry, University of Florida, Gainesville, FL 32611, USA"},{"name":"LinkedIn, Mountain View, Sunnyvale, CA 94043, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Trang T.","family":"Tran","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Florida, Gainesville, FL 32611, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Linh","family":"Pham","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Florida, Gainesville, FL 32611, USA"},{"name":"Department of Science and Mathematics, Texas A&amp;M University\u2014Central Texas, Killeen, TX 76549, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lingna","family":"Hu","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Florida, Gainesville, FL 32611, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kyle","family":"Bentz","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Florida, Gainesville, FL 32611, USA"},{"name":"Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9235-517X","authenticated-orcid":false,"given":"Daniel A.","family":"Savin","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Florida, Gainesville, FL 32611, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gail E.","family":"Fanucci","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Florida, Gainesville, FL 32611, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,11,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1580","DOI":"10.1126\/science.2420008","article-title":"HTLV-III gag protein is processed in yeast cells by the virus pol-protease","volume":"231","author":"Kramer","year":"1986","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"928","DOI":"10.1126\/science.2537531","article-title":"Molecular Modeling of the Hiv-1 Protease and Its Substrate Binding-Site","volume":"243","author":"Weber","year":"1989","journal-title":"Science"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"543","DOI":"10.1146\/annurev.bi.62.070193.002551","article-title":"Structure-Based Inhibitors of Hiv-1 Protease","volume":"62","author":"Wlodawer","year":"1993","journal-title":"Annu. 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