Democratizing single-molecule FRET for Dynamic Structural Biology: New tools and an integrative approach to understanding DNA dynamics Timothy D. Craggs, University of Sheffield Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between dynamic biomolecules. In the first part of this talk, I will present the smfBox1, a robust and economic confocal smFRET microscope constructed from readily available optical components, which is capable of accurate absolute FRET measurements (reproducing the FRET efficiencies from our multi-lab benchmarking study) and measuring biomolecular conformational dynamics (exemplified by DNA hairpin opening and closing) . I will then give an example of our iterative approach to determining conformational ensembles of biomolecules, in which we combined our smFRET experiments with molecular dynamic simulations to reveal the structure and dynamics of the DNA-protein complex formed between gapped-DNA and DNA Polymerase I. This led us to propose a general mechanism for substrate recognition by structure-specific DNA enzymes driven by protein sensing of the conformational dynamics of their substrates. Finally, I will present a new single-molecule method, which is sensitive to structural changes below 3 nm, Quantitative Quenchable FRET (qqFRET). This new tool extends the FRET toolbox for measurement of shorter distances, and also provides a single-molecule viscosity sensor, which we are beginning to exploit in studies of membrane fluidity and liquid-liquid phase separation. ***************************************************************************************************** Make sure to sign up to MeetAnyway before 3:30 pm so you have time to explore the networking area and discussion tables before the lecture starts. The recommended browser for this event is Google Chrome.
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