Semisynthetic protein nanoreactor for single-molecule chemistry

Area of Science:

• Biochemistry
• Nanotechnology
• Chemical Biology

Background:

• Protein pores can serve as nanoreactors for monitoring chemical reactions.
• Single-molecule analysis offers high sensitivity for studying reaction kinetics and intermediates.
• Unnatural amino acids can be incorporated into proteins to introduce novel functionalities.

Purpose of the Study:

• To develop a method for visualizing single-molecule click chemistry within a protein pore.
• To utilize the alpha-hemolysin (αHL) pore as a nanoreactor for covalent chemistry.
• To investigate reaction intermediates and kinetics at the single-molecule level.

Main Methods:

• Incorporation of an unnatural amino acid with a terminal alkyne group into the αHL pore via solid-phase peptide synthesis and native chemical ligation.
• Assembly of the heptameric αHL pore with one semisynthetic subunit.
• Monitoring ionic current flow through the pore to observe single-molecule reactions.

Main Results:

• Successful incorporation of an unnatural amino acid into the αHL pore.
• Visualization of click chemistry at the single-molecule level within the nanoreactor.
• Observation of a long-lived reaction intermediate (4.5 seconds).

Conclusions:

• The αHL pore can be effectively used as a nanoreactor for single-molecule covalent chemistry.
• The approach allows for the visualization of reaction intermediates and kinetics.
• This method provides a versatile platform for investigating a wide range of single-molecule chemical reactions.