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Mobile Molecules: Reactivity Profiling Guides Faster Movement on a Cysteine Track.

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Summary
This summary is machine-generated.

Researchers optimized a molecular hopper for faster biopolymer analysis. By tuning cysteine thiol reactivity within a protein nanopore, they significantly increased the hopping speed for enhanced characterization.

Keywords:
Mobile MoleculesNanoporesProtein EngineeringSingle-Molecule ChemistryThiol-Disulfide Interchange

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Area of Science:

  • Biophysics
  • Nanotechnology
  • Biochemistry

Background:

  • A molecular hopper utilizing thiol-disulfide exchange for sub-nanometer translocation within protein nanopores was previously developed.
  • The initial hopping rate was approximately 0.1 s-1, necessitating optimization for rapid, enzymeless biopolymer characterization.

Purpose of the Study:

  • To enhance the hopping rate of a molecular hopper for accelerated biopolymer translocation and characterization.
  • To investigate the reactivity profiles of individual cysteine footholds within a protein nanopore.
  • To identify and accelerate rate-limiting steps in the translocation process.

Main Methods:

  • Employed a single-molecule approach to determine the reactivity profiles of individual cysteine thiols.
  • Measured pKa values of cysteine thiols and pH-independent rate constants with a small-molecule disulfide.
  • Utilized site-specific mutagenesis and pH adjustments (from 8.5 to 9.5) to modify hopping dynamics.

Main Results:

  • Cysteine thiol pKa values within the nanopore ranged from 9.17 to 9.85.
  • Rate constants for thiolate reactions with disulfide varied up to 20-fold, indicating heterogeneous reactivity.
  • Increased pH and site-specific mutagenesis resulted in a 4-fold acceleration of the overall DNA cargo hopping rate.
  • The rate-limiting step of the translocation was accelerated 21-fold.

Conclusions:

  • Optimizing cysteine thiol reactivity through pH control and mutagenesis significantly enhances molecular hopper translocation speed.
  • Understanding individual foothold reactivity is crucial for engineering faster molecular machines.
  • This optimized system holds promise for rapid, enzymeless biopolymer characterization within nanopores.