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Updated: Feb 12, 2026

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules
Published on: April 25, 2025
Efficient use of single molecule time traces to resolve kinetic rates, models and uncertainties
Sonja Schmid1, Thorsten Hugel1
1Institute of Physical Chemistry II, University of Freiburg, Albertstr. 23 a, 79104 Freiburg, Germany.
Abstract:
Single molecule time traces reveal the time evolution of unsynchronized kinetic systems. Especially single molecule Förster resonance energy transfer (smFRET) provides access to enzymatically important time scales, combined with molecular distance resolution and minimal interference with the sample. Yet the kinetic analysis of smFRET time traces is complicated by experimental shortcomings-such as photo-bleaching and noise. Here we recapitulate the fundamental limits of single molecule fluorescence that render the classic, dwell-time based kinetic analysis unsuitable. In contrast, our Single Molecule Analysis of Complex Kinetic Sequences (SMACKS) considers every data point and combines the information of many short traces in one global kinetic rate model. We demonstrate the potential of SMACKS by resolving the small kinetic effects caused by different ionic strengths in the chaperone protein Hsp90. These results show an unexpected interrelation between conformational dynamics and ATPase activity in Hsp90.
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