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SCIENCE CHINA Chemistry, Volume 61, Issue 11: 1385-1388(2018) https://doi.org/10.1007/s11426-018-9231-2

Monitoring disulfide bonds making and breaking in biological nanopore at single molecule level

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  • ReceivedJan 5, 2018
  • AcceptedFeb 26, 2018
  • PublishedApr 24, 2018

Abstract

Monitoring subtle changes in ionic current flow through a nanopore could be applied to observe single molecule reaction. Here, we introduced cysteine to substitute for lysine at position 238 constructing a mutant aerolysin K238C. It could be regarded as a nanoreactor to efficiently visualize chemical bonds making and breaking. The compound 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was selected as a reactant coming into collisions on the interface of the pore to occur a reversible reaction. Our results showed that the mutant aerolysin could respond to three molecules of DTNB simultaneously and reflect corresponding levels with distinguishable current signals. Therefore, this method constitutes a simple, generic tool for monitoring single molecule reaction, which evokes a guidance for the mutant aerolysin towards the application of tracking other more reactions at single molecule level.


Funded by

the National Natural Science Foundation of China(21421004,21777041,21327807)

the Program of Introducing Talents of Discipline to Universities(B16017)

Innovation Program of Shanghai Municipal Education Commission(2017-01-07-00-02-E00023)

and the Fundamental Research Funds for the Central Universities(222201718001,222201717003)


Acknowledgment

This work was supported by the National Natural Science Foundation of China (21421004, 21777041, 21327807), the Program of Introducing Talents of Discipline to Universities (B16017), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-02-E00023), and the Fundamental Research Funds for the Central Universities (222201718001, 222201717003).


Interest statement

The authors declare that they have no conflict of interest.


Supplement

The supporting information is available online at chem.scichina.com and link.springer.com/journal/11426. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    Single-molecule reaction and structure of mutant aerolysin in the presence of DTNB. (a) A proposed reaction Cys-238 reacting with DTNB in a reversible reaction to form the mixed disulfide bonds. (b) A cross section of the aerolysin K238C with Cys at position 238 (red circularity). Cys was projected into the lumen of the pore to substitute for Lys. The two chambers were termed cis and trans, DTNB was added in the cis solution. (c) Top view of the β-barrel of mutant aerolysin K238C and seven Cys residues with red stick structure at the position 238. The exposed active Cys residues reacted with DTNB. All experiments were performed in the buffer of 1.0 M KCl, 1.0 mM EDTA and 10 mM Tris, at 24±1 °C, pH 8.0 (color online).

  • Figure 2

    Continuous current versus time traces with the addition of DTNB. (a) The β-barrel of wild-type aerolysin with Lys colored blue at the position 238 (left) and the current trace of wild-type aerolysin (right). DTNB traversed wild type aerolysin that did not produce blockade events. (b) The β-barrel of mutant aerolysin with Cys colored red at the position 238 (left) and the current trace of mutant aerolysin (right). DTNB traversed mutant aerolysin K238C that could react with the active sites in the lumen of a pore. The single-channel traces shown in the figures at +100 mV were filtered at 1 kHz. For display, (a) and (b) analyzed at Lowpass Bessel 100 Hz. The concentration of DTNB in the cis chamber is 30 μM. All experiments were performed in the buffer of 1.0 M KCl, 1.0 mM EDTA and 10 mM Tris, at 24±1 °C, pH 8.0 (color online).

  • Figure 3

    Reaction events and histograms of residual current at potential of +100 mV. (a) Reaction events at three levels corresponding to Level n (n=1, 2, 3). This was one distribution of molecules reacting with Cys residues shown in (a). (b) The histograms of Level 1, Level 2 and Level 3 fitted to Gaussian distribution. The fitted peaks were located at 47.90±0.06, 45.81±0.04 and 43.26±0.02 pA, respectively. The values of current for I0I1, I1I2, I2I3 were close, which means one level standing for single molecule reaction. All experiments were performed in the buffer of 1.0 M KCl, 1.0 mM EDTA and 10 mM Tris, at 24±1 °C, pH 8.0 (color online).

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