Insight into the mechanism of internalization of the cell-penetrating carrier peptide Pep-1 through conformational analysis

Biochemistry. 2004 Feb 17;43(6):1449-57. doi: 10.1021/bi035682s.


Recently, we described a new strategy for the delivery of proteins and peptides into mammalian cells, based on an amphipathic peptide of 21 residues, Pep-1, which was designed on the basis of a protein-interacting domain associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of active proteins or peptides both in cultured cells and in vivo, without requiring any covalent coupling. We have examined the conformational states of Pep-1 in its free form and complexed with a cargo peptide and have investigated their ability to interact with phospholipids and the structural consequences of these interactions. From the conformational point of view, Pep-1 behaves significantly differently from other similarly designed cell-penetrating peptides. CD analysis revealed a transition from a nonstructured to a helical conformation upon increase of the concentration. Determination of the structure by NMR showed that in water, its alpha-helical domain extends from residues 4-13. CD and FTIR indicate that Pep-1 adopts a helical conformation in the presence of phospholipids. Adsorption measurements performed at the air-water interface are consistent with the helical form. Pep-1 does not undergo conformational changes upon formation of a particle with a cargo peptide. In contrast, we observe a partial conformational transition when the complex encounters phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Conformational change of Pep-1 is not associated with complexation with its cargo but is induced upon association with the cell membrane.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Air
  • Amino Acid Sequence
  • Animals
  • Carrier Proteins / chemistry*
  • Carrier Proteins / metabolism
  • Cell Membrane Permeability*
  • Circular Dichroism
  • Detergents / chemistry
  • Female
  • Lipid Bilayers / chemistry
  • Models, Chemical
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Oocytes / metabolism
  • Peptides / chemistry*
  • Phospholipids / chemistry
  • Protein Binding
  • Protein Conformation
  • Protein Transport
  • Spectrometry, Fluorescence
  • Spectroscopy, Fourier Transform Infrared
  • Surface Properties
  • Water
  • Xenopus laevis


  • Carrier Proteins
  • Detergents
  • Lipid Bilayers
  • Peptides
  • Phospholipids
  • Water