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Penetratins 29
In a second set of experiments, the interaction of peptides with phospholipids
of various compositions and with SDS was characterized and the induced peptide
structure was determined. In the presence of SDS, penetratin adopts an α-helical
structure. 15,21-23 In SDS micelles ( 1 H-NMR with paramagnetic probes) penetratin-1
forms a straight helix. 22 The C terminus of the helix is deep in the SDS micelle and
its N terminus is near the surface of the micelle, with two residues (Arg 43 and
Gln 44) sticking outside.
Structural studies were also achieved with synthetic lipids. CD spectroscopy
showed that, although penetratin-1 adopts a random coil structure in an aqueous
environment, it becomes structured in the presence of negatively charged phospholipids.
At a low peptide to lipid ratio (1:325), the peptide adopts α-helical conformation.
18 At a high peptide to lipid ratio (1:10), the peptide forms antiparallel
β-sheet. 23 Indeed, Persson et al. have observed that above a 1:12.5 peptide to negatively
charged-lipid ratio, penetratin peptides induce vesicle aggregation, with concomitant
conformational transition from α-helix to antiparallel β-sheet. 24 Moreover,
following the vesicle aggregation process, they have monitored a spontaneous vesicle
disaggregation that may reflect penetratin translocation in phospholipid vesicles.
Another study on phospholipid monolayers with modulation infra-red reflection
absorption spectroscopy (PM-IRRAS) suggests that penetratin-1 is adsorbed parallel
to the surface of negatively charged phospholipid layers and adopts a β-sheet structure. 25
Since bilayer model membranes are more biomimetic than monolayers, Fragneto
et al. have analyzed the behavior of penetratin peptides in the bilayer model. 26,27 By
neutron and x-ray reflectivity, they were able to measure accurately bilayer structural
changes induced by penetratin peptides. They showed that penetratin added to a
bilayer of negatively charged phospholipids provokes more than a doubling of its
roughness and suggested that the peptide is mainly located in the lipid headgroups,
not in the acyl chains.
2.3.3 PROPOSED MODEL OF PENETRATIN INTERNALIZATION
In summary, a model can be proposed that takes into account all structure and
function and biophysical and biochemical studies on penetratin-1 and its derivatives.
17,28,29 These studies preclude classical endocytosis, fluid-phase potocytosis, and
pore formation. This last point recently has been addressed 30 and new results agree
with earlier data demonstrating that the addition of penetratin-1 to lipid monolayers
or cells does not induce pore formation (as evaluated by the passage of ions). Our
working hypothesis is that penetratin peptides interact directly with negatively
charged lipids or sugar components, presumably through an electrostatic interaction
and that this interaction is followed by destabilization of the lipid bilayer and
formation of inverted micelles in which the peptides are trapped (Figure 2.1). Eventually
a fraction of the micelles will open on the cytoplasmic side, thus allowing
intracellular delivery. In this model, penetratin is always kept in a hydrophilic
environment, included in the cavity of the micelle, and delivered from the extracellular
medium to the cytoplasm of the cell. This translocation mechanism led us to
speculate that hydrophilic molecules linked to penetratin peptides would also be
internalized.