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

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.