Leandro Cruz Rodríguez, Nahuel Naum Foressi, María Soledad Celej
{"title":"Tracking protein transitions through fluorescence spectral phasor analysis with ACDAN.","authors":"Leandro Cruz Rodríguez, Nahuel Naum Foressi, María Soledad Celej","doi":"10.1016/j.bpr.2025.100209","DOIUrl":null,"url":null,"abstract":"<p><p>This study investigates the use of spectral phasor analysis, hyperspectral imaging, and 6-acetyl-2-dimethylaminonaphthalene (ACDAN) fluorescence to explore key protein transitions: unfolding, amyloid aggregation, and liquid-liquid phase separation. We show that ACDAN fluorescence can sensitively detect subtle conformational changes before the complete protein unfolds, revealing early microenvironmental shifts. During amyloid formation, ACDAN identifies solvent dipolar relaxation events undetectable by conventional thioflavin T, providing critical insight into early aggregation events. Additionally, we map the physicochemical properties of protein biocondensates and highlight distinct microenvironments within these condensates, emphasizing the significance of dipolar relaxation in phase-separated systems. The approach provides a flexible and user-friendly toolkit for studying protein transitions, which can be easily implemented in commercial spectrofluorometers and microscopes.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"5 2","pages":"100209"},"PeriodicalIF":2.4000,"publicationDate":"2025-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biophysical reports","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.bpr.2025.100209","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOPHYSICS","Score":null,"Total":0}
引用次数: 0
Abstract
This study investigates the use of spectral phasor analysis, hyperspectral imaging, and 6-acetyl-2-dimethylaminonaphthalene (ACDAN) fluorescence to explore key protein transitions: unfolding, amyloid aggregation, and liquid-liquid phase separation. We show that ACDAN fluorescence can sensitively detect subtle conformational changes before the complete protein unfolds, revealing early microenvironmental shifts. During amyloid formation, ACDAN identifies solvent dipolar relaxation events undetectable by conventional thioflavin T, providing critical insight into early aggregation events. Additionally, we map the physicochemical properties of protein biocondensates and highlight distinct microenvironments within these condensates, emphasizing the significance of dipolar relaxation in phase-separated systems. The approach provides a flexible and user-friendly toolkit for studying protein transitions, which can be easily implemented in commercial spectrofluorometers and microscopes.
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