Dwell Time Analysis of a Single-Molecule Mechanochemical Reaction

Abstract

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.

Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

“…By measuring the rate of disulfide reduction by E. coli thioredoxin as a function of force on an engineered cysteine bond in a polypeptide chain, Wiita and coworkers were able to deduce sub-angstrom level conformation changes in the substrate during enzyme catalysis 138 . In similar experiments, Szoszkiewicz and coworkers examined the kinetics disulfide bond reduction using MFP combined with force clamp spectroscopy 139 . Another noteworthy achievement in the field of mechanochemistry is the work of Greene and coworkers 140 who showed that the protein kinase domains of Titin unfold in a step-wise manner, and speculated that kinases in muscle cells may be activated in response to the forces generated during each contraction/relaxation cycle, i.e.…”

Theoretical predictions of enforced structural changes in molecules

“…AFM techniques have also been used to enforce chemical reactions where both bond-breaking and bond formation take place [8,9] and to investigate the rupture of covalent bonds [10]. Scanning Tunneling Microscopy (STM) is another technique that has been used to form new chemical bonds.…”

Urinary Bladder-Relaxant Effect of Kurarinone Depending on Potentiation of Large-Conductance Ca2+-Activated K+ Channels

“…Then the mean amplitudes were plotted against the transmembrane voltage, and the slope conductance of the linear regression from each point was obtained. The distributions of the dwell times of the open and closed states recorded for single BK Ca channels were obtained in a linear histogram using Clampfit (Molecular Devices) as described in a previous study (Szoszkiewicz et al, 2008). The dwell-time distributions were fitted with a single-exponential function using simplex-least-squares fitting methods (Clampfit), and the exponential time-constant (mean open or closed time) was estimated.…”