Insight into the mechanism of internalization of the cell-penetrating carrier peptide Pep-1 through conformational analysis.
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.
Deshayes
Sébastien
S
CRBM-CNRS, FRE 2593, 1919 route de Mende, F-34293 Montpellier Cedex, France.
Heitz
Annie
A
Morris
May C
MC
Charnet
Pierre
P
Divita
Gilles
G
Heitz
Frédéric
F
D000388:Air; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002463:Cell Membrane Permeability; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D005260:Female; D008051:Lipid Bilayers / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013499:Surface Properties; D014867:Water; D014982:Xenopus laevis
D002352:Carrier Proteins; D003902:Detergents; D008051:Lipid Bilayers; D010455:Peptides; D010743:Phospholipids; D014867:Water