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Here, single\u2010molecule localization microscopy, polarization\u2010dependent single\u2010molecule 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\u00b0 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.<\/jats:p>","DOI":"10.1002\/smtd.202402114","type":"journal-article","created":{"date-parts":[[2025,2,3]],"date-time":"2025-02-03T02:25:53Z","timestamp":1738549553000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Monitoring Dynamic Conformations of a Single Fluorescent Molecule Inside a Protein Cavity"],"prefix":"10.1002","volume":"9","author":[{"given":"Santiago","family":"Sosa","sequence":"first","affiliation":[{"name":"Centro de Investigaciones en Bionanociencias (CIBION) Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET)  Godoy Cruz 2390 Ciudad Aut\u00f3noma de Buenos Aires C1425FQD Argentina"},{"name":"Fundaci\u00f3n Instituto Leloir IIBBA\u2010CONICET  Av. Patricias Argentinas 435 Ciudad Aut\u00f3noma de Buenos Aires C1405BWE Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3795-3230","authenticated-orcid":false,"given":"Alan M.","family":"Szalai","sequence":"additional","affiliation":[{"name":"Centro de Investigaciones en Bionanociencias (CIBION) Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET)  Godoy Cruz 2390 Ciudad Aut\u00f3noma de Buenos Aires C1425FQD Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luc\u00eda F.","family":"Lopez","sequence":"additional","affiliation":[{"name":"Centro de Investigaciones en Bionanociencias (CIBION) Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET)  Godoy Cruz 2390 Ciudad Aut\u00f3noma de Buenos Aires C1425FQD Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juan Manuel","family":"Prieto","sequence":"additional","affiliation":[{"name":"Instituto de Qu\u00edmica Biol\u00f3gica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN)\u2010CONICET Facultad de Ciencias Exactas y Naturales Universidad de Buenos Aires  G\u00fciraldes 2620 Ciudad Aut\u00f3noma de Buenos Aires C1428EGA Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cecilia","family":"Zaza","sequence":"additional","affiliation":[{"name":"Centro de Investigaciones en Bionanociencias (CIBION) Consejo Nacional de Investigaciones Cient\u00edficas y T\u00e9cnicas (CONICET)  Godoy Cruz 2390 Ciudad Aut\u00f3noma de Buenos Aires C1425FQD Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aleksandra K.","family":"Adamczyk","sequence":"additional","affiliation":[{"name":"Department of Physics University of Fribourg  Chemin du Mus\u00e9e 3 Fribourg CH\u20101700 Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hern\u00e1n R.","family":"Bonomi","sequence":"additional","affiliation":[{"name":"Fundaci\u00f3n Instituto Leloir IIBBA\u2010CONICET  Av. 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