Single-Molecule Analysis beyond Dwell Times: Demonstration and Assessment in and out of Equilibrium

Sonja Schmid, Markus Götz, Thorsten Hugel*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

42 Citations (Scopus)


We present a simple and robust technique for extracting kinetic rate models and thermodynamic quantities from single-molecule time traces. Single-molecule analysis of complex kinetic sequences (SMACKS) is a maximum-likelihood approach that resolves all statistically relevant rates and also their uncertainties. This is achieved by optimizing one global kinetic model based on the complete data set while allowing for experimental variations between individual trajectories. In contrast to dwell-time analysis, which is the current standard method, SMACKS includes every experimental data point, not only dwell times. As a result, it works as well for long trajectories as for an equivalent set of short ones. In addition, the previous systematic overestimation of fast over slow rates is solved. We demonstrate the power of SMACKS on the kinetics of the multidomain protein Hsp90 measured by single-molecule Förster resonance energy transfer. Experiments in and out of equilibrium are analyzed and compared to simulations, shedding new light on the role of Hsp90’s ATPase function. SMACKS resolves accurate rate models even if states cause indistinguishable signals. Thereby, it pushes the boundaries of single-molecule kinetics beyond those of current methods.

Original languageEnglish
Pages (from-to)1375-1384
Number of pages10
JournalBiophysical Journal
Issue number7
Publication statusPublished - 4 Oct 2016
Externally publishedYes


Dive into the research topics of 'Single-Molecule Analysis beyond Dwell Times: Demonstration and Assessment in and out of Equilibrium'. Together they form a unique fingerprint.

Cite this