Short talk:
FRET measurements resolving structures, dynamics and transitions of various biomolecules

Julian Folz1, Suren Felekyan1, Ralf Kühnemuth1, Claus A.M. Seidel1

1Heinrich Heine University Düsseldorf, Düsseldorf, Germany

Fluorescence spectroscopy and imaging are important biophysical techniques to study biomolecules in vitro. The use of more than one fluorophore per molecule opens additional opportunities arising from photon densities, coincidences and dipolar coupling by Förster Resonance Energy Transfer (FRET) to study the stoichiometry, structure, dynamics and transitions of biomolecular systems. This phenomenon we applied to a wide range of biomolecules with a varying size from 100 kDa to 1400 kDa showing different dynamics and kinetics on timescales from μs to hours. To study this we combine established methods like Multiparameter Fluorescence Detection (MFD) of freely diffusing molecules and Total Internal Reflection (TIRF) microscopy using immobilized molecules, together with new approaches pushing the collection efficiency to detection signals to hundreds of kHz. The outcome are observables describing (a) different structural states and dynamics of the human Guanalyte Binding Protein (hGBP1) and its behavior in the farnesylated state, (b) the live exchange of different molecular states of a freely diffusing 4-way junction on a μs timescale and (c) the structural behavior of a Tc toxin on a ms-timescale giving insights into fast/straight forward and complex/distributed transitions crucial for its biological functionality.

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