Spontaneous intermolecular amide bond formation between side chains for irreversible peptide targeting

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Peptides are crucial for biological analysis but suffer from unstable interactions.
• Limited surface area and flexibility hinder peptide-protein binding stability.
• Existing peptide tags are small, minimizing protein function perturbation.

Purpose of the Study:

• To design a peptide tag that forms a stable, irreversible amide bond with a protein partner.
• To leverage intramolecular amide bond formation from pilin subunits for this purpose.
• To enable robust protein labeling and modification.

Main Methods:

• Engineered a 16-amino acid peptide derived from Streptococcus pyogenes pilin.
• Utilized spontaneous amide bond formation between lysine and asparagine side chains.
• Tested reaction efficiency, pH/temperature dependence, and buffer compatibility.
• Assessed in vitro and in vivo (E. coli, mammalian cells) performance.

Main Results:

• Achieved 98% conversion in amide bond formation between peptide and protein partners.
• Reaction occurred efficiently across pH 5-8, in various buffers, and independently of redox state.
• Observed similar reaction rates at 4°C and 37°C.
• Demonstrated efficient reconstitution in E. coli and specific surface labeling on mammalian cells.

Conclusions:

• Developed a peptide tag enabling irreversible protein targeting through amide bond formation.
• The tag is stable, efficient, and functional in diverse biological conditions and systems.
• Potential applications include bioassembly, cellular imaging, and stabilizing protein complexes under force.