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68 Cell-Penetrating Peptides: Processes and Applications
The molecular modeling of interaction of transportan and its analogues yields
structures that correspond well to NMR data on the peptide in SDS micelles. The
C-terminal part of the transportan analogues is predicted to be buried in the membrane;
the only exception is TP15, whose N terminus becomes buried in the membrane.
Both Lys residues of C terminus of the longest peptides (TP, TP7, and TP9)
may reach the opposite side of the membrane, while the aromatic residues of the N
terminal and the biotinyl group remain anchored in the first phospholipid polar head
area.
According to calculation of the molecular hydrophobic potentials (MHP), 37 the
N-terminal part is hydrophilic due to the presence of threonine, serine, or aromatic
residues. Strikingly, TP15 has an inverted orientation, although this is the sole peptide
that has a charged hydrophilic lysine residue instead of Lys-Ile-Leu in the C terminus.
Thus, the most important features that determine the orientation of cell penetrating
peptides in the membrane are peptide length and location of aromatic and lysine
residues in the terminal regions of the sequence.
The transportan analogues have stable positions when inserted into the membrane,
but their orientations in relation to the membrane surface differ. Therefore,
we tried to group the results according to angle and depth of penetration into the
membrane, a classification tightly correlated to peptide size. Two predicted parameters
may be correlated with the ability of the peptides to traverse phospholipid
membranes. First, an oblique orientation relative to the membrane plane seems to
be important, since peptides predicted to have a more parallel orientation in relation
to the membrane (angle less than 40 to 45°) internalize efficiently. In contrast, short
peptides (22 amino acids or less) with more perpendicular orientation internalize
poorly (e.g., TP 11), if at all (TP 13 and TP 15). Second, transportan and its
derivatives of 24–25 amino acids, i.e., long analogues, penetrate into cells more
efficiently. The increased length of the peptide seems to facilitate penetration into
the hydrophobic core. Shorter peptides (less than 21 residues) may not reach the
second leaflet and are therefore too short to span the lipid bilayer.
Although transportan is in random-coil formation when water solution, it seems
to be organized as an ensemble of different multimers, ranging from predominant
dimers to molecular assemblies with high molecular weight. Multimerization can
be induced by the interaction of peptide with detergent micelles or membrane lipids,
as suggested by Derossi et al. 1 However, when we used biotinyl–transportan to
transduce the tagged streptavidin into cells, we often observed formation of large,
labeled rod-like structures in both electron and fluorescence microscopy, probably
generated due to multimerization of transportan in solution. 38 Huge assemblies form
preferentially when streptavidin is incubated with biotinyl–transportan in a small
volume, for a long time, or at high concentration, lending more support to the
probable multimerization of transportan in solution. Both penetratin and transportan
tend to form multimers and assemblies in solution; whether this is coincident or
somehow related to their ability to penetrate into cells is not clear yet.
We have demonstrated that transportan, a chimera consisting of galanin (1–13)
coupled to mastoparan (1–14) is a cell-penetrating peptide. Furthermore, the uptake
of transportan is probably not receptor or energy dependent. Transportan can be
shortened by at least six amino acids and modified with respect to its amphiphilicity,