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The interaction of partially unfolded proteins with drugs remains poorly understood due to a lack of structural information on proteins in their partially unfolded states. Here, we studied the interaction between serum albumin (bovine serum albumin (BSA) as a model system), an abundant protein in blood serum that is an effective carrier for numerous known drugs, and a nonsteroidal anti-inflammatory drug (NSAID) naproxen (NPS) using various spectroscopic and computational methods. Molecular dynamics simulations starting from the drug-unbound state and performed at physiological and higher temperatures revealed novel hydrophobic sites on the BSA surface. We analyzed the BSA\u2013NPS interaction in the presence and absence of the cationic organized assembly CTAB and two oligosaccharides (\u03b2-CD and 2-HP-\u03b2-CD) at different excitation wavelengths. The solvation dynamics of BSA under NPS-bound conditions became \u223c4.6% faster. Oligosaccharides were found to increase the solubility of NPS by providing a hydrophobic environment for the formation of inclusion complexes through host\u2013guest interactions. These findings provide a comprehensive overview and uncover the binding model and mechanism of interaction of NPS with BSA, revealing hydrophobic and electrostatic interactions and hydrogen bonds required for BSA to bind NPS at these noncanonical sites. The molecular-level understanding of the binding mechanism of commonly used NSAIDs like NPS with partially unfolded BSA will be useful in designing pharmaceutically important molecules with efficient loading and delivery properties.<\/jats:p>","DOI":"10.1021\/acs.jpcb.4c03901","type":"journal-article","created":{"date-parts":[[2024,9,24]],"date-time":"2024-09-24T14:35:08Z","timestamp":1727188508000},"page":"9327-9340","source":"Crossref","is-referenced-by-count":10,"title":["Drug Binding to Partially Unfolded Serum Albumin:\nInsights into Nonsteroidal Anti-Inflammatory Drug Naproxen\u2013BSA\nInteractions from Spectroscopic and MD Simulation Studies"],"prefix":"10.1021","volume":"128","author":[{"given":"Debasish","family":"Rout","sequence":"first","affiliation":[{"name":"Indian Institute of Technology Jodhpur , , , ,","place":["Jodhpur, RJ, India, 342037"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arun Kumar","family":"Upadhyaya","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology Jodhpur , , , ,","place":["Jodhpur, RJ, India, 342037"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pratibha","family":"Agarwala","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology Jodhpur , , , ,","place":["Jodhpur, RJ, India, 342037"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chanchal","family":"Sharma","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology Jodhpur , , , ,","place":["Jodhpur, RJ, India, 342037"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arumay","family":"Pal","sequence":"additional","affiliation":[{"name":"Vellore Institute of Technology , , ,","place":["Bhopal, India, 466114"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4148-5326","authenticated-orcid":true,"given":"Dibyendu K.","family":"Sasmal","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology Jodhpur , , , ,","place":["Jodhpur, RJ, India, 342037"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2024,9,24]]},"reference":[{"key":"2026081805150026500_cit1","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1186\/1556-276X-9-343","article-title":"Bovine serum albumin\nnanoparticles as controlled release carrier for local drug delivery\nto the inner ear","volume":"9","author":"Yu","year":"2014","journal-title":"Nanoscale Res. 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Hematol.\nOncol."},{"key":"2026081805150026500_cit8","doi-asserted-by":"publisher","first-page":"421","DOI":"10.1210\/me.15.3.421","article-title":"Probing conformational changes in\nthe estrogen receptor:\nevidence for a partially unfolded intermediate facilitating ligand\nbinding and release","volume":"15","author":"Gee","year":"2001","journal-title":"Mol. Endocrinol."},{"issue":"2","key":"2026081805150026500_cit9","doi-asserted-by":"publisher","first-page":"452","DOI":"10.1016\/j.jmb.2007.07.060","article-title":"A conformational\nunfolding reaction activates phage\nfd for the infection of Escherichia coli","volume":"373","author":"Eckert","year":"2007","journal-title":"J.\nMol. 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Environ. Chem. Eng."},{"issue":"1","key":"2026081805150026500_cit19","doi-asserted-by":"publisher","first-page":"102","DOI":"10.1345\/aph.10423","article-title":"Clinical pharmacology\nand pharmacokinetics of amprenavir","volume":"36","author":"Sadler","year":"2002","journal-title":"Annals of\nPharmacotherapy"},{"issue":"1","key":"2026081805150026500_cit20","doi-asserted-by":"publisher","first-page":"82","DOI":"10.1021\/acs.jmedchem.5b00909","article-title":"Structural insights into the competitive binding of\ndiclofenac and naproxen by equine serum albumin","volume":"59","author":"Sekula","year":"2016","journal-title":"Journal of medicinal chemistry"},{"key":"2026081805150026500_cit21","doi-asserted-by":"publisher","DOI":"10.1016\/j.colsurfb.2024.113841","article-title":"Covalent modification\nof \u03b3-cyclodextrin with geraniol: An antibacterial agent with\ngood thermal stability, solubility and biocompatibility","volume":"237","author":"Ding","year":"2024","journal-title":"Colloids Surf., B"},{"issue":"10","key":"2026081805150026500_cit22","doi-asserted-by":"publisher","first-page":"281","DOI":"10.3390\/ph13100281","article-title":"Cyclic oligosaccharides\nas active drugs, an updated\nreview","volume":"13","author":"Matencio","year":"2020","journal-title":"Pharmaceuticals"},{"key":"2026081805150026500_cit23","doi-asserted-by":"publisher","first-page":"199","DOI":"10.1002\/9781119865452.ch8","article-title":"Biomedical\nApplications\nof Cyclodextrin","author":"Ahmad","year":"2024","journal-title":"Biopolymers for Biomedical\nApplications"},{"key":"2026081805150026500_cit24","doi-asserted-by":"publisher","first-page":"14759","DOI":"10.1039\/D3CC04048H","article-title":"New Opportunities\nof Cyclodextrins in Supramolecular\nAssembly: Metal Organic Framework, Crystalline self-Assembly, and\nCatalyzed Assembly","volume":"59","author":"Yan","year":"2023","journal-title":"Chem. 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Chem."},{"issue":"1","key":"2026081805150026500_cit28","doi-asserted-by":"publisher","first-page":"5","DOI":"10.1016\/j.jphotochem.2006.11.011","article-title":"Effect of the structure and concentration\nof cyclodextrins in the quenching process of naproxen","volume":"188","author":"Valero","year":"2007","journal-title":"J. Photochem. Photobiol., A"},{"issue":"2","key":"2026081805150026500_cit29","doi-asserted-by":"publisher","first-page":"155","DOI":"10.5599\/jese.205","article-title":"Spectroscopic,\nvoltammetry and molecular docking study of binding interaction of\nantipsychotic drug with bovine serum albumin","volume":"6","author":"Mahanthappa","year":"2016","journal-title":"J. Electrochem. Sci. Eng."},{"key":"2026081805150026500_cit30","doi-asserted-by":"publisher","first-page":"67","DOI":"10.1016\/j.saa.2012.12.007","article-title":"Binding of\nnaproxen enantiomers to human serum albumin studied by fluorescence\nand room-temperature phosphorescence","volume":"105","author":"Lammers","year":"2013","journal-title":"Spectrochimica\nActa Part A: Molecular and Biomolecular Spectroscopy"},{"issue":"18","key":"2026081805150026500_cit31","doi-asserted-by":"publisher","DOI":"10.1002\/cphc.202200265","article-title":"Molecular\nMechanism of Interaction\nof Curcumin with BSA, Surfactants and Live E. Coli Cell Membrane Revealed\nby Fluorescence Spectroscopy and Confocal Microscopy","volume":"23","author":"Agarwala","year":"2022","journal-title":"ChemPhysChem"},{"issue":"64","key":"2026081805150026500_cit32","doi-asserted-by":"publisher","first-page":"38873","DOI":"10.1039\/D0RA06323A","article-title":"Multi-spectroscopic monitoring of\nmolecular interactions between an amino acid-functionalized ionic\nliquid and potential anti-Alzheimer\u2019s drugs","volume":"10","author":"Sharma","year":"2020","journal-title":"RSC Adv."},{"key":"2026081805150026500_cit33","doi-asserted-by":"crossref","DOI":"10.1007\/978-0-387-46312-4","volume-title":"Principles of fluorescence\nspectroscopy","author":"Lakowicz","year":"2006"},{"issue":"4","key":"2026081805150026500_cit34","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1007\/s12039-022-02092-8","article-title":"Monitoring\nthe effect of SDS on the solvation dynamics and structural conformation\nof \u03b2-casein","volume":"134","author":"Mubashira","year":"2022","journal-title":"J. Chem. Sci."},{"issue":"3","key":"2026081805150026500_cit35","doi-asserted-by":"publisher","first-page":"1095","DOI":"10.1021\/la504270a","article-title":"Hydrophobicity\nis the governing factor\nin the interaction of human serum albumin with bile salts","volume":"31","author":"Ghosh","year":"2015","journal-title":"Langmuir"},{"issue":"16","key":"2026081805150026500_cit36","doi-asserted-by":"publisher","first-page":"3397","DOI":"10.1021\/acs.jpcb.9b00219","article-title":"Structure,\nactivity, and dynamics of human serum albumin\nin a crowded pluronic F127 hydrogel","volume":"123","author":"Nandy","year":"2019","journal-title":"J. Phys.\nChem. 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Pharmaceutics"},{"issue":"47","key":"2026081805150026500_cit40","doi-asserted-by":"publisher","first-page":"24147","DOI":"10.1021\/jp062734p","article-title":"Binding of\nnaproxen and amitriptyline\nto bovine serum albumin: biophysical aspects","volume":"110","author":"Banerjee","year":"2006","journal-title":"J. Phys. Chem. B"},{"issue":"5","key":"2026081805150026500_cit41","doi-asserted-by":"publisher","first-page":"2501","DOI":"10.1021\/acs.molpharmaceut.4c00044","article-title":"Role for Carboxylic\nAcid Moiety in NSAIDs: Favoring the Binding at Site II of Bovine Serum\nAlbumin","volume":"21","author":"de\nCarvalho Bertozo","year":"2024","journal-title":"Mol. Pharmaceutics"},{"issue":"11","key":"2026081805150026500_cit42","doi-asserted-by":"publisher","first-page":"4385","DOI":"10.1039\/C8NJ06229C","article-title":"A diboronic acid fluorescent\nsensor for selective recognition of d-ribose via fluorescence quenching","volume":"43","author":"Wang","year":"2019","journal-title":"New J. Chem."},{"key":"2026081805150026500_cit43","doi-asserted-by":"publisher","first-page":"126","DOI":"10.1016\/j.jphotobiol.2016.11.016","article-title":"Synthesis, characterization\nand serum albumin binding\nstudies of vitamin K3 derivatives","volume":"166","author":"Suganthi","year":"2017","journal-title":"Journal of\nPhotochemistry and Photobiology B: Biology"},{"issue":"3","key":"2026081805150026500_cit44","doi-asserted-by":"publisher","first-page":"148","DOI":"10.1016\/j.jpha.2016.10.001","article-title":"Multi-spectroscopic\ncharacterization of bovine serum\nalbumin upon interaction with atomoxetine","volume":"7","author":"Buddanavar","year":"2017","journal-title":"Journal\nof pharmaceutical analysis"},{"key":"2026081805150026500_cit45","doi-asserted-by":"publisher","first-page":"211","DOI":"10.4236\/ajac.2014.53026","article-title":"Optimization\nof quantitative analysis of naproxin sodium using UV spectrophotometery\nin different solvent mediums","volume":"5","author":"Zuberi","year":"2014","journal-title":"Am. J. Anal. Chem."},{"issue":"3","key":"2026081805150026500_cit46","doi-asserted-by":"publisher","first-page":"484","DOI":"10.1016\/j.jpba.2011.06.003","article-title":"Characterization and relative response factor determination\nof process related impurity in Naproxen by nuclear magnetic resonance\nspectroscopy","volume":"56","author":"Iqbal","year":"2011","journal-title":"J. Pharm. Biomed. Anal."},{"issue":"7","key":"2026081805150026500_cit47","doi-asserted-by":"publisher","first-page":"5676","DOI":"10.1039\/C5NJ00785B","article-title":"Interaction of a novel platinum drug\nwith bovine serum albumin: FTIR and UV-Vis spectroscopy analysis","volume":"39","author":"Korkmaz","year":"2015","journal-title":"New J. Chem."},{"issue":"12","key":"2026081805150026500_cit48","doi-asserted-by":"publisher","first-page":"124909","DOI":"10.1063\/1.2178782","article-title":"Temperature\ndependence of solvation dynamics and anisotropy decay in a protein:\nANS in bovine serum albumin","volume":"124","author":"Sahu","year":"2006","journal-title":"J. Chem. Phys."},{"issue":"4","key":"2026081805150026500_cit49","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1007\/s12039-022-02092-8","article-title":"Monitoring\nthe effect of SDS on the solvation dynamics and structural conformation\nof \u03b2-casein","volume":"134","author":"Mubashira","year":"2022","journal-title":"J. Chem. Sci."},{"issue":"4","key":"2026081805150026500_cit50","doi-asserted-by":"publisher","first-page":"477","DOI":"10.1016\/j.jtusci.2015.01.010","article-title":"Investigations of mixed surfactant\nsystems of lauryl alcohol-based\nbis sulfosuccinate anionic gemini surfactants with conventional surfactants:\nA fluorometric study","volume":"9","author":"Singh","year":"2015","journal-title":"Journal of Taibah University\nfor Science"},{"issue":"13","key":"2026081805150026500_cit51","doi-asserted-by":"publisher","first-page":"6847","DOI":"10.1021\/acs.jmedchem.0c00225","article-title":"Albumin-Based Transport of Nonsteroidal\nAnti-Inflammatory\nDrugs in Mammalian Blood Plasma","volume":"63","author":"Mateusz","year":"2020","journal-title":"J. Med. Chem."},{"issue":"2","key":"2026081805150026500_cit52","doi-asserted-by":"publisher","first-page":"429","DOI":"10.1016\/j.jconrel.2011.11.028","article-title":"Clinical Impact of\nSerum Proteins on Drug Delivery","volume":"161","author":"Kratz","year":"2012","journal-title":"J. Controlled\nRelease"},{"issue":"4","key":"2026081805150026500_cit53","doi-asserted-by":"publisher","first-page":"728","DOI":"10.3390\/pharmaceutics14040728","volume":"14","author":"Cho","year":"2022","journal-title":"Pharmaceutics."},{"issue":"1","key":"2026081805150026500_cit54","doi-asserted-by":"publisher","first-page":"38","DOI":"10.1016\/j.jmb.2005.07.075","article-title":"Structural Basis of\nthe Drug-Binding\nSpecificity of Human Serum Albumin","volume":"353","author":"Ghuman","year":"2005","journal-title":"J. Mol.\nBiol."}],"container-title":["The Journal of Physical Chemistry B"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jpcb.4c03901","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jpcbfk\/article-pdf\/128\/39\/9327\/14964969\/jp4c03901.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jpcbfk\/article-pdf\/128\/39\/9327\/14964969\/jp4c03901.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T10:15:35Z","timestamp":1787048135000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jpcbfk\/article\/128\/39\/9327\/1423651\/Drug-Binding-to-Partially-Unfolded-Serum-Albumin"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,9,24]]},"references-count":54,"journal-issue":{"issue":"39","published-online":{"date-parts":[[2024,9,24]]},"published-print":{"date-parts":[[2024,10,3]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jpcb.4c03901","relation":{},"ISSN":["1520-6106","1520-5207"],"issn-type":[{"value":"1520-6106","type":"print"},{"value":"1520-5207","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,9,24]]}}}