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Biomolecules can have multiple sites or mode of interaction with other molecular entities, so that a resolution of the individual binding events in terms of spatial localization as well as association and dissociation kinetics is required for a meaningful description. Here we describe a trichromatic fluorescent binding\u2010 and displacement assay for simultaneous monitoring of three individual binding sites in the important transporter and binding protein human serum albumin. Independent investigations of binding events by X\u2010ray crystallography and time\u2010resolved dynamics measurements (switchSENSE technology) confirm the validity of the assay, the localization of binding sites and furthermore reveal conformational changes associated with ligand binding. The described assay system allows for the detailed characterization of albumin\u2010binding drugs and is therefore well\u2010suited for prediction of drug\u2010drug and drug\u2010food interactions. Moreover, conformational changes, usually associated with binding events, can also be analyzed.<\/jats:p>","DOI":"10.1002\/cmdc.202000069","type":"journal-article","created":{"date-parts":[[2020,3,12]],"date-time":"2020-03-12T00:00:48Z","timestamp":1583971248000},"page":"738-743","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["Resolving Binding Events on the Multifunctional Human Serum Albumin"],"prefix":"10.1002","volume":"15","author":[{"given":"Lea","family":"Wenskowsky","sequence":"first","affiliation":[{"name":"Institute of Organic Chemistry Johannes Gutenberg-University  Duesbergweg 10\u201314 55128 Mainz Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael","family":"Wagner","sequence":"additional","affiliation":[{"name":"Sanofi-Aventis Deutschland GmbH Industriepark H\u00f6chst  65926 Frankfurt am Main Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Johannes","family":"Reusch","sequence":"additional","affiliation":[{"name":"Dynamic Biosensors GmbH Lochhamer Stra\u00dfe 15  82152 Martinsried\/Planegg Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Herman","family":"Schreuder","sequence":"additional","affiliation":[{"name":"Sanofi-Aventis Deutschland GmbH Industriepark H\u00f6chst  65926 Frankfurt am Main Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hans","family":"Matter","sequence":"additional","affiliation":[{"name":"Sanofi-Aventis Deutschland GmbH Industriepark H\u00f6chst  65926 Frankfurt am Main Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Till","family":"Opatz","sequence":"additional","affiliation":[{"name":"Institute of Organic Chemistry Johannes Gutenberg-University  Duesbergweg 10\u201314 55128 Mainz Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5596-8543","authenticated-orcid":false,"given":"Stefan Matthias","family":"Petry","sequence":"additional","affiliation":[{"name":"Sanofi-Aventis Deutschland GmbH Industriepark H\u00f6chst  65926 Frankfurt am Main Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2020,3,19]]},"reference":[{"key":"e_1_2_2_1_1","unstructured":"\u00a0"},{"key":"e_1_2_2_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/sj.bjp.0707251"},{"key":"e_1_2_2_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/0006-2952(81)90151-9"},{"key":"e_1_2_2_4_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1749-6632.1973.tb20486.x"},{"key":"e_1_2_2_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2275(20)36723-7"},{"key":"e_1_2_2_6_2","doi-asserted-by":"publisher","DOI":"10.1080\/15216540500404093"},{"key":"e_1_2_2_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.mam.2011.12.002"},{"key":"e_1_2_2_8_1","unstructured":"\u00a0"},{"key":"e_1_2_2_9_2","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.2000.4158"},{"key":"e_1_2_2_10_2","doi-asserted-by":"publisher","DOI":"10.1038\/1869"},{"key":"e_1_2_2_11_2","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.2000.5208"},{"key":"e_1_2_2_12_2","doi-asserted-by":"publisher","DOI":"10.2133\/dmpk.24.342"},{"key":"e_1_2_2_13_1","doi-asserted-by":"publisher","DOI":"10.1002\/ange.201710437"},{"key":"e_1_2_2_14_1","unstructured":"\u00a0"},{"key":"e_1_2_2_15_2","doi-asserted-by":"publisher","DOI":"10.1172\/JCI103742"},{"key":"e_1_2_2_16_2","volume-title":"All about albumin: biochemistry, genetics, and medical applications","author":"Peters T.","year":"1995"},{"key":"e_1_2_2_17_1","first-page":"113","volume":"9","author":"Job P.","year":"1928","journal-title":"Ann. 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