Linear prediction and single-channel recording

Carter, Alison A.; Oswald, Robert E.

doi:10.1016/0165-0270(94)00221-2

Cited by 3 publications

(4 citation statements)

References 15 publications

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“…Under what circumstances would any other transition state become distinguishable in the forceclamp experiments? Sine et al, 22 Carter et al, 43 and Wachtel et al 44 have approached the problem of resolving different transition states. Taking into account double-exponential kinetics with two mean rates r and r′ of equal amplitude, they could still resolve only cases with r/r′ > 2.5.…”

Section: Comparisons Between the Analysis Results For Disulfidementioning

confidence: 99%

Dwell Time Analysis of a Single-Molecule Mechanochemical Reaction

et al. 2007

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Force-clamp spectroscopy is a novel technique for studying mechanochemistry at the single-bond level. Single disulfide bond reduction events are accurately detected as stepwise increases in the length of polyproteins that contain disulfide bonds and that are stretched at a constant force with the cantilever of an atomic force microscope (AFM). The kinetics of this reaction has been measured from single-exponential fits to ensemble averages of the reduction events. However, exponential fits are notoriously ambiguous to use in cases of kinetic data showing multiple reaction pathways. Here we introduce a dwell time analysis technique, of widespread use in the single ion channel field, that we apply to the examination of the kinetics of reduction of disulfide bonds measured from single-molecule force-clamp spectroscopy traces. In this technique, exponentially distributed dwell time data is plotted as a histogram with a logarithmic time scale and a square root ordinate. The advantage of logarithmic histograms is that exponentially distributed dwell times appear as well-defined peaks in the distribution, greatly enhancing our ability to detect multiple kinetic pathways. We apply this technique to examine the distribution of dwell times of 4488 single disulfide bond reduction events measured in the presence of two very different kinds of reducing agents: tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) and the enzyme thioredoxin (TRX). A different clamping force is used for each reducing agent to obtain distributions of dwell times on a similar time scale. In the case of TCEP, the logarithmic histogram of dwell times showed a single peak, corresponding to a single reaction mechanism. By contrast, similar experiments done with TRX showed two well-separated peaks, marking two distinct modes of chemical reduction operating simultaneously. These experiments demonstrate that dwell time analysis techniques are a powerful approach to studying chemical reactions at the single-molecule level.

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Section: Comparisons Between the Analysis Results For Disulfidementioning

confidence: 99%

Dwell Time Analysis of a Single-Molecule Mechanochemical Reaction

et al. 2007

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“…Next, a given clamping force was set for 60 s to collect all the reduction events, and not the outliers only. Disulfide bond cleavage was not observed in the absence of hTrx 16 . Any significant changes in the protein end-to-end length occur after the molecule is extended by at least tens of nanometers.…”

Section: Proteins Andmentioning

confidence: 82%

“…Theoretical and experimental single molecule studies of disulfide reductions by small inorganic molecules obtained that a direct mechanism, e.g., a S N 2 mechanism, would produce strongly force-accelerated disulfide reductions with Δx of 30 -40 pm, i.e., corresponding to 20 % stretching of a -S-S-bond from its initial length of 205 pm to 240 pm at a transition state 16,26 . However, in recent FC-AFM experiments a transition from Δx ~ 40 -50 pm to Δx ~ 5 -15 pm was observed above 500 pN clamping force 27 .…”

Section: Discussionmentioning

confidence: 99%

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Single-molecule studies of disulfide bond reduction pathways used by human thioredoxin

Szoszkiewicz

2013

Biophysical Chemistry

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How to cite this manuscriptIf you make reference to this version of the manuscript, use the following information: Szoszkiewicz, R. (2013). Single-molecule studies of disulfide bond reduction pathways used by human thioredoxin. Retrieved from http://krex.ksu.edu Published Version InformationCitation: Szoszkiewicz, R. (2013 8 molecules are stretched at constant force applied by a cantilever in a force-clamp mode of atomic force microscopy (FC-AFM). Disulfide reduction events are accurately detected from stepwise increases in the end-to-end length of (I27 SS ) 8 . Earlier FC-AFM studies observed one disulfide reduction pathway used by hTrx and suggested an additional electron tunnelling mechanism. Here, a very large set of unbiased FC-AFM data is collected in a range of clamping forces. By analyzing the data using exponential fits and dwell times histograms two disulfide reduction pathways used by hTrx are resolved. Based on previous studies one of these pathways is attributed to force-dependent Michaelis-Menten catalysis. The latter reduction pathway is weakly forceinhibited and occurs sporadically. Bimolecular nucleophilic substitutions (S N 2) and electron tunnelling (ET) mechanisms are discussed to explain the second pathway. Direct S N 2 and ET mechanisms cannot be discounted, but a hypothetical E2-S N 2 mechanism involving a hydride reducing a disulfide bond provides an interesting alternative, which needs to be verified in future experiments.

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Force-Extension and Force-Clamp AFM Spectroscopies in Investigating Mechanochemical Reactions and Mechanical Properties of Single Biomolecules

Szoszkiewicz

2009

Scanning Probe Microscopy in Nanoscience and Nanotechnology

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Linear prediction and single-channel recording

Cited by 3 publications

References 15 publications

Dwell Time Analysis of a Single-Molecule Mechanochemical Reaction

Dwell Time Analysis of a Single-Molecule Mechanochemical Reaction

Single-molecule studies of disulfide bond reduction pathways used by human thioredoxin

Force-Extension and Force-Clamp AFM Spectroscopies in Investigating Mechanochemical Reactions and Mechanical Properties of Single Biomolecules

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