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206 Cell-Penetrating Peptides: Processes and Applications
to the axis of the barrel. In Figure 9.6B, the 3D envelope of the restraints shows a
local minimum at –40° for orientation of the β-sheet and a mass center penetration
at about –7 Å. The mass center is shifted because of all the hydrophilic loops at the
extremity of the pore of each monomer which move into an aqueous environment.
Validation of the method now allows testing ab initio or homology models of
membrane proteins of unknown structures, to try to authenticate the insertion and
structure of the model within a lipid environment.
9.3.2 PENETRATIN
Penetratins are cell-permeant peptides that can carry various sorts of cargoes into
live cells and hence are now considered as vectors to develop new therapeutic tools.
However, the mechanisms of penetratin cellular entry remain unknown. To get insight
into the process of its membrane crossing and to provide a framework for further
design of new derivatives, we have used the Monte Carlo method to model the
penetratin membrane interactions.
For this aim, different analyses have been performed on:
Antennapedia homeodomain in complex with a double-stranded DNA cognate
site (AntHD–DNA complex)
Antennapedia homeodomain alone (AntHD)
First helix of AntHD (RYQTLELEKEFHF)
Second helix of AntHD (RRRRIEIAHAL)
Third helix of AntHD corresponding to the penetratin sequence (pAntp;
RQIKIWFQNRRMKWKK)
The AntHD–DNA complex structure, 9ent, has been determined by x-ray and
NMR analysis. 66 The complex is represented in Figure 9.7A with each of the AntHD
helices. For sake of clarity, hydrogen atoms are not represented but are accounted
for in subsequent analysis. The 9ent structure and three homeodomain helices have
been imported in the HyperChem V6 (Hypercube, Inc., Florida) software, and the
AMBER force field has allowed us to attribute the atomic charges (Figure 9.7B).
The AntHD–DNA complex is highly negatively charged due to the phosphate
groups of the double-strand DNA. The sum of the atomic charges, obtained from
the AMBER force field, leads to a total charge of –18.23, whereas the AntHD peptide
in the absence of the DNA sequence has a total charge of +11.76.
The first homeodomain helix (Figure 9.7B, top) contains three negatively
charged (Glu), one positively charged (Arg), and two polar residues (Tyr, His).
Nevertheless, according to the AMBER force field, its total charge is equal to zero.
The second homeodomain helix (Figure 9.7B, middle) has five charged residues:
four arginines at its N-terminal, a glutamic acid, and a polar amino acid (His). Its
global charge is about +4.00. Among the 16 amino-acid residues of the penetratin
sequence (Figure 9.7B, bottom), only four are hydrophobic. The others are charged
or polar, conferring to this helix a global charge of +6.76.
If calculation of atomic charges gives information about the polarity of the
Antennapedia homeodomain and its constitutive α-helices, calculation of molecular