De Novo Designed Cell-Penetrating Peptide Self-Assembly Featuring Distinctive Tertiary Structure

Area of Science:

• Biochemistry
• Biophysics
• Drug Delivery

Background:

• Protein design advancements parallel biopharmaceutical development.
• Protein quaternary structures, like oligomers, are crucial for function.
• Cell penetration of peptides is vital for drug delivery, but oligomer instability hinders study.

Purpose of the Study:

• To characterize oligomerization states of amphipathic peptide LK-3.
• To investigate the functional role of LK-3 oligomers in cell penetration.
• To explore the impact of enantiomeric mixtures on peptide oligomerization and cell penetration.

Main Methods:

• X-ray crystallography for atomic structure determination.
• Förster resonance energy transfer (FRET) to assess oligomerization and cell penetration.
• Native mass spectrometry (MS) to confirm higher-order oligomers.
• Differential scanning calorimetry (DSC) to analyze thermal stability.

Main Results:

• LK-3 forms stable hexamers (trimer of dimers) with a hydrophobic core.
• Oligomer formation of LK-3 correlates with enhanced cell penetration.
• A 1:1 mixture of L/D-peptide dimers showed superior cell penetration compared to pure enantiomers.
• Enantiomeric mixtures promote functional oligomer formation.

Conclusions:

• Amphipathic peptide LK-3 forms functional oligomers at low nanomolar concentrations.
• Oligomerization is a key determinant of LK-3's cell penetration efficacy.
• Enantiomeric peptide mixtures offer a strategy to enhance cell-penetrating peptide function.