{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T14:52:20Z","timestamp":1786978340033,"version":"build-2736575974"},"reference-count":41,"publisher":"Wiley","issue":"12","license":[{"start":{"date-parts":[[2009,10,1]],"date-time":"2009-10-01T00:00:00Z","timestamp":1254355200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"name":"Intramural Research Program of the NIDDK, National Institutes of Health"},{"name":"National Science Foundation","award":["MCB 0814905"],"award-info":[{"award-number":["MCB 0814905"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2009,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Purification and\n                    <jats:italic>in vitro<\/jats:italic>\n                    protein\u2010folding schemes were developed to produce monodisperse samples of the mature wild\u2010type HIV\u20102 protease (PR2), enabling a comprehensive set of biochemical and biophysical studies to assess the dissociation of the dimeric protease. An E37K substitution in PR2 significantly retards autoproteolytic cleavage during expression. Furthermore, it permits convenient measurement of the dimer dissociation of PR2\n                    <jats:sub>E37K<\/jats:sub>\n                    (elevated\n                    <jats:italic>K<\/jats:italic>\n                    <jats:sub>d<\/jats:sub>\n                    \u223c20 n\n                    <jats:italic>M<\/jats:italic>\n                    ) by enzyme kinetics. Differential scanning calorimetry reveals a\n                    <jats:italic>T<\/jats:italic>\n                    <jats:sub>m<\/jats:sub>\n                    of 60.5 for PR2 as compared with 65.7\u00b0C for HIV\u20101 protease (PR1). Consistent with weaker binding of the clinical inhibitor darunavir (DRV) to PR2, the\n                    <jats:italic>T<\/jats:italic>\n                    <jats:sub>m<\/jats:sub>\n                    of PR2 increases by 14.8\u00b0C in the presence of DRV as compared with 22.4\u00b0C for PR1. Dimer interface mutations, such as a T26A substitution in the active site (PR2\n                    <jats:sub>T26A<\/jats:sub>\n                    ) or a deletion of the C\u2010terminal residues 96\u201399 (PR2\n                    <jats:sub>1\u201395<\/jats:sub>\n                    ), drastically increase the\n                    <jats:italic>K<\/jats:italic>\n                    <jats:sub>d<\/jats:sub>\n                    (&gt;10\n                    <jats:sup>5<\/jats:sup>\n                    \u2010fold). PR2\n                    <jats:sub>T26A<\/jats:sub>\n                    and PR2\n                    <jats:sub>1\u201395<\/jats:sub>\n                    consist predominantly of folded monomers, as determined by nuclear magnetic resonance (NMR) and size\u2010exclusion chromatography coupled with multiangle light scattering and refractive index measurements (SMR), whereas wild\u2010type PR2 and its active\u2010site mutant PR2\n                    <jats:sub>D25N<\/jats:sub>\n                    are folded dimers. Addition of twofold excess active\u2010site inhibitor promotes dimerization of PR2\n                    <jats:sub>T26A<\/jats:sub>\n                    but not of PR2\n                    <jats:sub>1\u201395<\/jats:sub>\n                    , indicating that subunit interactions involving the C\u2010terminal residues are crucial for dimer formation. Use of SMR and NMR with PR2 facilitates probing for potential inhibitors that restrict protein folding and\/or dimerization and, thus, may provide insights for the future design of inhibitors to circumvent drug resistance.\n                  <\/jats:p>","DOI":"10.1002\/pro.261","type":"journal-article","created":{"date-parts":[[2009,10,1]],"date-time":"2009-10-01T17:19:52Z","timestamp":1254417592000},"page":"2442-2453","source":"Crossref","is-referenced-by-count":24,"title":["Revealing the dimer dissociation and existence of a folded monomer of the mature HIV\u20102 protease"],"prefix":"10.1002","volume":"18","author":[{"given":"John 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