Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Schmid S

PROVIDER: S-EPMC5052450 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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

Schmid Sonja S Götz Markus M Hugel Thorsten T

Biophysical journal 20161001 7

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 ...[more]

PMID: 27705761

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Project description:The acetylcholine receptor (AChR) from vertebrate skeletal muscle initiates voluntary movement, and its kinetics of activation are crucial for maintaining the safety margin for neuromuscular transmission. Furthermore, the kinetic mechanism of the muscle AChR serves as an archetype for understanding activation mechanisms of related receptors from the Cys-loop superfamily. Here we record currents through single muscle AChR channels with improved temporal resolution approaching half an order of magnitude over our previous best. A range of concentrations of full and partial agonists are used to elicit currents from human wild-type and gain-of-function mutant AChRs. For each agonist-receptor combination, rate constants are estimated from maximum likelihood analysis using a kinetic scheme comprised of agonist binding, priming, and channel gating steps. The kinetic scheme and rate constants are tested by stochastic simulation, followed by incorporation of the experimental step response, sampling rate, background noise, and filter bandwidth. Analyses of the simulated data confirm all rate constants except those for channel gating, which are overestimated because of the established effect of noise on the briefest dwell times. Estimates of the gating rate constants were obtained through iterative simulation followed by kinetic fitting. The results reveal that the agonist association rate constants are independent of agonist occupancy but depend on receptor state, whereas those for agonist dissociation depend on occupancy but not on state. The priming rate and equilibrium constants increase with successive agonist occupancy, and for a full agonist, the forward rate constant increases more than the equilibrium constant; for a partial agonist, the forward rate and equilibrium constants increase equally. The gating rate and equilibrium constants also increase with successive agonist occupancy, but unlike priming, the equilibrium constants increase more than the forward rate constants. As observed for a full and a partial agonist, the gain-of-function mutation affects the relationship between rate and equilibrium constants for priming but not for channel gating. Thus, resolving brief single channel currents distinguishes priming from gating steps and reveals how the corresponding rate and equilibrium constants depend on agonist occupancy.

Dataset Information

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

Publications

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

Similar Datasets

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