Efficient use of single molecule time traces to resolve kinetic rates, models and uncertainties

TL;DR

This paper introduces SMACKS, a new method for analyzing single molecule FRET data that overcomes traditional limitations, enabling detailed kinetic modeling and revealing new insights into Hsp90 protein dynamics.

Contribution

The paper presents SMACKS, a novel analysis approach that utilizes all data points from multiple short traces to accurately determine kinetic rates in single molecule experiments.

Findings

SMACKS effectively resolves small kinetic effects in Hsp90.

Reveals unexpected link between conformational dynamics and ATPase activity.

Overcomes limitations of classic dwell-time analysis.

Abstract

Single molecule time traces reveal the time evolution of unsynchronized kinetic systems. Especially single molecule F\"orster resonance energy transfer (smFRET) provides access to enzymatically important timescales, 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.