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

SOSA, SANTIAGO; SZALAI, ALAN M.; LOPEZ, LUCÍA F.; PRIETO, JUAN MANUEL; ZAZA, CECILIA; ADAMCZYK, ALEKSANDRA K.; BONOMI, HERNÁN R.; MARTI, MARCELO A.; ACUNA, GUILLERMO P.; GOLDBAUM, FERNANDO A.; STEFANI, FERNANDO D.

Small Methods

Wiley

Año: 2025

2366-9608

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 bonds inside a protein cavity. The observed conformations are in good agreement with molecular dynamics simulations, enabling a new, more realistic interplay between experiments and simulations to identify stable conformations and the key interactions involved. Furthermore, jumps between conformations can be monitored with a precision of 3° and a time resolution of a few seconds, confirming the potential of this methodology for retrieving dynamic structural information of nanoscopic biological systems under physiologically compatible conditions.