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

Area of Science:

• Biophysics
• Nanotechnology
• Biochemistry

Background:

• A molecular hopper utilizing thiol-disulfide chemistry for sub-nanometer steps within a protein nanopore was previously developed.
• The initial hopping rate of approximately 0.1 s^-1 limits applications in rapid, enzymeless biopolymer characterization.

Purpose of the Study:

• To optimize the hopping rate of the molecular hopper for enhanced speed in biopolymer translocation.
• To investigate the reactivity of individual cysteine footholds and identify rate-limiting steps for targeted improvement.

Main Methods:

• Employed a single-molecule approach to determine the reactivity profiles and pK_a values of individual cysteine thiols within the nanopore.
• Utilized site-specific mutagenesis and pH adjustments (from 8.5 to 9.5) to modulate hopping dynamics.
• Quantified the impact of these modifications on the translocation rate of a DNA cargo.

Main Results:

• Cysteine thiol pK_a values within the pore ranged from 9.17 to 9.85.
• pH-independent rate constants for thiolate reactions with a small-molecule disulfide showed up to a 20-fold variation.
• The overall hopping rate of DNA cargo was accelerated 4-fold, and the rate-limiting step was accelerated 21-fold through mutagenesis and pH increase.

Conclusions:

• Optimization of cysteine foothold reactivity and environmental pH significantly enhances molecular hopper translocation speed.
• This improved molecular hopper system shows promise for rapid, enzymeless biopolymer characterization within nanopores.
• Understanding individual foothold kinetics is crucial for efficient molecular machine design and performance tuning.