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Small Methods2025;9(8):e2402114.August 1, 2025Journal Article

Monitoring Dynamic Conformations of a Single Fluorescent Molecule Inside a Protein Cavity.

Santiago Sosa1,2, Alan M Szalai1, Lucía F Lopez1, Juan Manuel Prieto3, Cecilia Zaza1, Aleksandra K Adamczyk4, Hernán R Bonomi2,5, Marcelo A Marti3,6, Guillermo P Acuna4,7, Fernando A Goldbaum8, Fernando D Stefani1,9
1Centro de Investigaciones en Bionanociencias (CIBION), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2390, Ciudad Autónoma de Buenos Aires, C1425FQD, Argentina.
2Fundación Instituto Leloir, IIBBA-CONICET, Av. Patricias Argentinas 435, Ciudad Autónoma de Buenos Aires, C1405BWE, Argentina.
3Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN)-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Güiraldes 2620, Ciudad Autónoma de Buenos Aires, C1428EGA, Argentina.
4Department of Physics, University of Fribourg, Chemin du Musée 3, Fribourg, CH-1700, Switzerland.
5Generate Biomedicines, Somerville, MA, 02143, USA.
6Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Güiraldes 2620, Ciudad Autónoma de Buenos Aires, C1428EGA, Argentina.
7Swiss National Center for Competence in Research (NCCR) Bio-inspired Materials, University of Fribourg, Chemin des Verdiers 4, Fribourg, CH-1700, Switzerland.
8Centro de Rediseño e Ingeniería de Proteínas and Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín (CRIP-IIB-UNSAM), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Campus Miguelete, Buenos Aires, CP1650, Argentina.
9Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Güiraldes 2620, Ciudad Autónoma de Buenos Aires, C1428EHA, Argentina.
PubMed DOI

Abstract

Fluorescence nanoscopy and single-molecule methods are entering the realm of structural biology, breaking new ground for dynamic structural measurements at room temperature and liquid environments. Here, single-molecule localization microscopy, polarization-dependent single-molecule excitation, and protein engineering are combined to determine the orientation of a fluorophore forming hydrogen bond…

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