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Resolving the Dynamic Non-Covalent Interaction inside Membrane Protein Channel by Single-Molecule Interaction Spectrum

preprint
submitted on 25.10.2018, 09:41 and posted on 25.10.2018, 13:27 by Meng-Yin Li, Yi-Lun Ying, Xi-Xin Fu, Jie Yu, Shao-Chuang Liu, Ya-Qian Wang, Shuang Li, Chan Cao, Yong-Jing Wan, Yi-Tao Long
Millions of years of evolution have produced membrane protein channels capable of efficiently moving ions across the cell membrane. The underlying fundamental mechanisms that facilitate these actions greatly contribute to the weak non-covalent interactions. However, uncovering these dynamic interactions and its synergic network effects still remains challenging in both experimental techniques and molecule dynamics (MD) simulations. Here, we present a rational strategy that combines MD simulations and frequency-energy spectroscopy to identify and quantify the role of non-covalent interactions in carrier transport through membrane protein channels, as encoded in traditional single channel recording or ionic current. We employed wild-type aerolysin transporting of methylcytosine and cytosine as a model to explore the dynamic ionic signatures with non-stationary and non-linear frequency analysis. Our data illuminate that methylcytosine experiences strong non-covalent interactions with the aerolysin nanopore at Region 1 around R220 than cytosine, which produces characteristic frequency-energy spectra. Furthermore, we experimentally validate the obtained hypothesis from frequency-energy spectra by designing single-site mutation of K238G which creates significantly enhanced non-covalent interactions for the recognition of methylcytosine. The frequency-energy spectrum of ions flowing inside membrane channels constitutes a single-molecule interaction spectrum, which bridges the gap between traditional ionic current recording and the MD simulations, facilitating the qualitative and quantitive description of the non-covalent interactions inside membrane channels.

Funding

This research was supported by the National Natural Science Foundation of China (21834001 and 61871183), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-02-E00023) and the Fundamental Research Funds for the Central Universities (222201718001, 222201717003).

History

Email Address of Submitting Author

ytlong@ecust.edu.cn

Institution

East China University of Science and Technology

Country

China

ORCID For Submitting Author

0000-0003-2571-7457

Declaration of Conflict of Interest

The authors declare no competing financial interests.

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