Abstract
Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Forster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90 degrees intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.
| Dokumententyp: | Zeitschriftenartikel |
|---|---|
| EU Funded Grant Agreement Number: | 638536 |
| EU-Projekte: | Horizon 2020 > ERC Grants > ERC Starting Grant > ERC Grant 638536: SM-IMPORT - Substrate import at work: single-molecule studies of ABC transporters |
| Fakultät: | Chemie und Pharmazie > Department Chemie |
| Themengebiete: | 500 Naturwissenschaften und Mathematik > 540 Chemie |
| ISSN: | 1520-6106 |
| Sprache: | Englisch |
| Dokumenten ID: | 48198 |
| Datum der Veröffentlichung auf Open Access LMU: | 27. Apr. 2018, 08:14 |
| Letzte Änderungen: | 26. Okt. 2021, 12:04 |
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