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BP: Fachverband Biologische Physik

BP 26: Single molecules biophysics

BP 26.3: Vortrag

Donnerstag, 4. April 2019, 15:45–16:00, H4

Angstrom precision distance measurements in dynamic protein structures with single-molecule FRET — •Christian Gebhardt, Rebecca Mächtel, Niels Zijlstra, and Thorben Cordes — LMU München, Faculty of Biology, Großhaderner Str. 2-4, 82152 Planegg, Germany

Single-molecule Förster resonance energy transfer (smFRET) has evolved towards a mature toolkit for the study of distances, structures and dynamics of biomolecules in a physiologically relevant context in vitro and in vivo. There is, however, no generally accepted way to derive and use quantitative distance information from the FRET-ruler to derive structural models or constraints in the protein data base. Hellenkamp et al. (Nat. Methods, 2018) recently presented a quantitative smFRET study of oligonucleotide ruler structures that revealed high precision, accuracy and reproducibility of FRET-derived distances in a worldwide comparative study of 20 labs with a distance uncertainty below 6 angstrom. While this establishes smFRET as a suitable technique for accurate distance measurements of static biological reference structures, we raise the question if smFRET is applicable for proteins with dynamic conformational motions or allosteric modulations of protein structure by an effector. Proteins are more challenging targets for site-specific fluorophore labelling. We identified a model system to benchmark FRET-derived distance uncertainties in proteins for situations of (i) stochastic labelling and (ii) allosteric and dynamic modulation of the structure and show similar angstrom precision comparable to DNA.