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This\u00a0study\u00a0introduces a protocol for identifying binding sites and characterizing ligand coordination modes in situ by combining fluorescence spectroscopy with efficient atomistic simulations based on the TrESP\u2011MMPol model. The protocol integrates electrostatic potential\u2011fitted transition charges with a polarizable classical environment, thereby overcoming the orientation and dielectric\u2010screening assumptions inherent to F\u00f6rster theory. The protocol has been applied to human serum albumin (HSA) and a library of fluorescent small molecules, including known binders of the HSA, accurately reproducing the binding sites of naproxen, carprofen, and indomethacin, and revealing novel binding scenarios for other molecules. The results show that direct comparison of experimental FRET data with atomistically simulated observables enables discrimination of plausible binding models \u2013 including the site and binding mode \u2013 and avoids systematic errors in distance estimation. The protocol is particularly attractive to examine targets with a single tryptophan, and can also be extended to other targets of interest in drug discovery via site\u2010labelling with unnatural amino acids.<\/jats:p>","DOI":"10.1002\/smtd.202501820","type":"journal-article","created":{"date-parts":[[2025,11,14]],"date-time":"2025-11-14T14:26:16Z","timestamp":1763130376000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Characterization of Ligand Binding in Human Serum Albumin from Atomistic Energy Transfer Simulations"],"prefix":"10.1002","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2340-7279","authenticated-orcid":false,"given":"\u00d6zge","family":"Erg\u00fcn","sequence":"first","affiliation":[{"name":"Departament de Farm\u00e0cia i Tecnologia Farmac\u00e8utica i Fisicoqu\u00edmica Facultat de Farm\u00e0cia i Ci\u00e8ncies de l'Alimentaci\u00f3 Universitat de Barcelona (UB)  Barcelona 08028 Spain"},{"name":"Institut de Qu\u00edmica Te\u00f2rica i Computacional (IQTCUB) Universitat de Barcelona (UB)  Barcelona 08028 Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7971-6540","authenticated-orcid":false,"given":"Andrea","family":"Bertran\u2010Mostazo","sequence":"additional","affiliation":[{"name":"Departament de Farm\u00e0cia i Tecnologia Farmac\u00e8utica i Fisicoqu\u00edmica Facultat de Farm\u00e0cia i Ci\u00e8ncies de l'Alimentaci\u00f3 Universitat de Barcelona (UB)  Barcelona 08028 Spain"},{"name":"Institut de Biomedicina (IBUB) Universitat de Barcelona (UB)  Barcelona 08028 Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2239-6670","authenticated-orcid":false,"given":"Elena","family":"Cubero","sequence":"additional","affiliation":[{"name":"Gain Therapeutics Sucursal en Espa\u00f1a  Barcelona Science Park Barcelona 08028 Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1702-0369","authenticated-orcid":false,"given":"Carles","family":"Galdeano","sequence":"additional","affiliation":[{"name":"Departament de Farm\u00e0cia i Tecnologia Farmac\u00e8utica i Fisicoqu\u00edmica Facultat de Farm\u00e0cia i Ci\u00e8ncies de l'Alimentaci\u00f3 Universitat de Barcelona (UB)  Barcelona 08028 Spain"},{"name":"Institut de Biomedicina (IBUB) Universitat de Barcelona (UB)  Barcelona 08028 Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0070-1208","authenticated-orcid":false,"given":"Carles","family":"Curutchet","sequence":"additional","affiliation":[{"name":"Departament de Farm\u00e0cia i Tecnologia Farmac\u00e8utica i Fisicoqu\u00edmica Facultat de Farm\u00e0cia i Ci\u00e8ncies de l'Alimentaci\u00f3 Universitat de Barcelona (UB)  Barcelona 08028 Spain"},{"name":"Institut de Qu\u00edmica Te\u00f2rica i Computacional (IQTCUB) Universitat de Barcelona (UB)  Barcelona 08028 Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,11,14]]},"reference":[{"key":"e_1_2_8_1_1","volume-title":"Protein Fluorescence","author":"Lakowicz J. 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