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Controlling Nanopore Dynamics via Loop Stapling and Unstapling for Tunable Substrate Transport.

Arya Krishna1, Neethu Puthumadathil1, Dheeraj Kumar Sarkar2

  • 1Transdisciplinary Research Program, BRIC-Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695014, India.

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Summary

Researchers engineered CymA nanopores for biosensing by controlling N-terminus flexibility. This allows dual-mode sensing of small peptides and large cyclic sugars, advancing nanopore sensor design.

Keywords:
current blockagescyclic sugarsloop dynamicitynanoporeporinssingle-channel

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Area of Science:

  • Biophysics
  • Nanotechnology
  • Biochemistry

Background:

  • Biological nanopores are key tools in nanopore biosensing.
  • CymA, a membrane porin, has a unique structure with a constricted N-terminus that regulates substrate transport.
  • The dynamic role of CymA's N-terminus is not well understood.

Purpose of the Study:

  • Investigate CymA conformational dynamics using protein engineering, electrical recordings, and molecular dynamics simulations.
  • Understand the role of the N-terminus in regulating molecular transport.
  • Develop advanced CymA nanopores for enhanced biosensing applications.

Main Methods:

  • Protein engineering to create a "stapled CymA" mutant with a disulfide-bonded N-terminus.
  • Electrical recordings to monitor ion flow and analyte translocation.
  • Molecular dynamics simulations to analyze conformational changes.

Main Results:

  • Stapled CymA restricted large cyclic sugar translocation but allowed small peptide passage.
  • Disrupting the disulfide bond restored N-terminus flexibility and cyclic sugar translocation.
  • Engineered CymA with a dynamic, untethered N-terminus minimized gating for effective single-molecule sensing.

Conclusions:

  • N-terminus flexibility is a tunable element for regulating molecular transport in nanopores.
  • Conformational control of CymA enables dual-mode sensing of diverse analytes.
  • This work provides a strategy for designing dynamic nanopores for advanced biosensing.