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

• DOI: 10.1002/smtd.202402114
• Publication Date: 2025-02-03T07:25:53Z
• Authors: Santiago Sosa; Alan M. Szalai; Lucía F. Lopez; Juan Manuel Prieto; Cecilia Zaza; Aleksandra K. Ada...; Hernán R. Bonomi; Marcelo A. Marti; Guillermo P. Acuna; Fernando A. Goldbaum; Fernando D. Stefani

Abstract Fluorescence nanoscopy and single‐molecule methods are entering the realm of structural biology, breaking new ground for dynamic measurements at room temperature liquid environments. Here, localization microscopy, polarization‐dependent excitation, protein engineering combined to determine orientation a fluorophore forming hydrogen bonds inside cavity. The observed conformations in good agreement with molecular dynamics simulations, enabling new, more realistic interplay between experiments simulations identify stable key interactions involved. Furthermore, jumps can be monitored precision 3° time resolution few seconds, confirming potential this methodology retrieving information nanoscopic biological systems under physiologically compatible conditions.