Dwell time analysis of a single-molecule mechanochemical reaction.
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.
Szoszkiewicz
Robert
R
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Ainavarapu
Sri Rama Koti
SR
Wiita
Arun P
AP
Perez-Jimenez
Raul
R
Sanchez-Ruiz
Jose M
JM
Fernandez
Julio M
JM
D004220:Disulfides / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics; D010720:Phosphines / Q000737:chemistry*; D011487:Protein Conformation; D013057:Spectrum Analysis / Q000295:instrumentation / Q000379:methods; D013314:Stress, Mechanical; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics; D013997:Time Factors
D004220:Disulfides; D009124:Muscle Proteins; D010720:Phosphines; D013879:Thioredoxins; C080938:tris(2-carboxyethyl)phosphine