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210 Cell-Penetrating Peptides: Processes and Applications
translocation across the bilayer, we applied the Monte Carlo procedure to each helix
separately. The energy restraint evolution as a function of the mass center penetration
shows that no helix can cross the uncharged bilayer model. However, the pAntp
interacts more closely with the phospholipid polar heads (Figure 9.8E) than the
second helix (Figure 9.8D), which adsorbs to the membrane, and than the first one
(Figure 9.8C), which almost does not interact with the polar heads.
In conclusion, these Monte Carlo simulations performed with an uncharged
bilayer model clearly show that no Antennapedia-derived peptide is predicted to
cross the membrane. These results are in agreement with the MHP analysis: the
charged and polar residues within the three homeodomain helices make them particularly
hydrophilic so that they cannot enter the hydrophobic core of a biological
membrane.
9.3.2.2 Charged Bilayer Model
Biological membranes are rarely symmetrical in lipid and protein composition. The
uncharged model of lipid bilayer used in the first round of simulations described
earlier mimics a membrane made of only neutral phospholipids (PL°). However, if
the major membrane components of the eukaryotic organisms are PL°, it has been
shown 67 that negatively charged phospholipids (PL – ) also enter in the composition
of membranes. These PL – are asymmetrically distributed between both leaflets of
membranes. Moreover, the asymmetric distribution of PL° and PL – is different
according to the cell type and is actively controlled by specific protein machineries.
The asymmetric presence of PL – causes an electrostatic gradient in biological
membranes. To take this into account, we introduced charges in both leaflets. The
question then was to determine to which extent the force field produced by the
charge asymmetry might influence passage of peptides through the model membrane.
In order to start such simulations, we chose to set up a model of the human
erythrocyte membrane. Cullis et al. 67 have calculated a superficial charge density of
8 µC/cm 2 (0.005 e – /Å 2 ) for the erythrocyte membrane. This charge density is due to
the presence of PL – in the internal leaflet of the erythrocyte membrane. We performed
new simulations, considering that the internal sheet of the bilayer had a charge
density ranging from 0.005 e – /Å 2 to 0.04 e – /Å 2 .
The results of the different simulations are presented in Table 9.2. According to
these simulations, penetratin should cross a membrane when the superficial charge
density is 0.01 e – /Å 2 . When the superficial charge density reaches 0.04 e – /Å 2 , the
Antennapedia homeodomain, as well as the first helix, partially penetrates the membrane,
while the second helix and the penetratin cross this membrane. We detailed
two simulations in order to further explain the mechanism.
Figure 9.9A shows evolution of the hydrophobic, lipid perturbation (E lip), and
charge sum restraints as a function of mass center penetration of the AntHD–DNA,
when considering a superficial charge density of 0.02 e – /Å 2 in the inner leaflet. The
two dotted lines delimit the phospholipid polar heads of the external leaflet. The
bilayer center is defined by the 0 Å ordinate. Because the complex does not cross
the membrane, only the outer leaflet bearing the charge was plotted.