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10.02.15 10:56

Award | Thomas Peulen

By: M. Froescher

Thomas Peulen wins the poster award of the Fluorescence Subgroup at the Biophysical Society Meeting in Baltimore, USA.

Titel: Positional Fluorophore Properties in High-Precision FRET Analysis: Orientation Effects, Dynamic Quenching and Beyond

Abstract:

To understand the function of biomolecules experimental techniques with high spatial and temporal resolution are essential. The characteristic time of global protein kinetics is in the order of microseconds or slower. FRET-measurements are ideally suited to study biomolecular function, as conformation dynamics can be monitored with high temporal resolution from nanoseconds to the observation time. FRET is considered a low resolution technique providing limited structural information.

This is caused as: (1) Measurements are performed in solution causing averaging of distances (2) In intensity based FRET the spatial resolution in dynamic systems is limited the photon count-rate (3) The rate constant of energy transfer dependents on the relative orientation of the dipoles (4) The dyes are coupled to the biomolecule by flexible linkers resulting in distribution of distances limiting the application of conventional modelling tools (5) The interactions of dyes with the biomolecule change the fluorescence properties by quenching of their excited states.

We present solutions to these problems gathered in a software-suite. Averaging of distances is overcome by analysis of the intensity on the nanosecond time scale, preferentially recoded using an multiparameter fluorescence detection setup. Uncertainties regarding the dipole orientation are minimized by anisotropy measurements. Flurophore distributions are calculated according to the FRET-Positioning System [1]. Dye-protein interactions are treated by global analysis of the time-resolved donor fluorescence in absence and in presence of the acceptor. A visual approach facilitates the analysis of time-resolved fluorescence. This approach is validated by experiments and Monte-Carlo simulations of the flurophores capturing dynamic quenching, orientation effects and unspecific interaction with the biomolecular surface. Application examples for several proteins studied by single-molecule and ensemble high-precision FRET measurements are given.

[1] Kalinin et al. Nature Methods 9, 1218-1225 (2012)

 

 

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