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Biophysical Studies of Cell-Penetrating Peptides 237
pores in the form of peptide complexes. The peptide activities result in membrane
leakage (permeation) and consecutive translocation of the peptide as well as the
lipids in a transient manner. 47,48 This picture seems to be valid for peptides like
melittin, magainin, and mastoparan X.
It has been suggested that each peptide has two physical states of binding to a
phospholipid bilayer. 33,52,53 At low peptide-to-lipid molar ratios, the peptide becomes
adsorbed in the membrane head-group region. In this state the peptide is still
functionally inactive, but then causes thinning of the membrane bilayer. Above a
threshold value for the molar ratio, the peptide forms a multiple-pore state. This
lethal state depends on the composition and biophysical nature of the membrane,
which could explain the susceptibility and specificity of the biological activity.
With buforin 2 peptide (from toad stomach) no membrane pores were detected,
in agreement with the low lytic effect. 54 Interestingly, buforin efficiently translocates
across lipid bilayers, but in this case without membrane permeabilization and lipid
flip-flop induction. Its antimicrobial activity is even higher then for the pore-forming
peptides. The toxic effect of buforin is suggested to be an interaction with intracellular
nucleic acids.
The uptake of labeled buforin into Escherichia coli cells could be detected by
fluorescence microscopy. However, the bacteria cells were not permeabilized to allow
an influx of a DNA-staining probe. The cell-penetrating efficiency and antimicrobial
activity of buforin and its analogs correlated with their α-helical contents. 55 It seems
as if buforin in its behavior may have some properties in common with CPPs. Hence,
it would be interesting to test whether buforin, or any of its analogs, can in fact pull
a cargo into a cell as efficiently as some (arche)typical CPPs.
Molecular details of the membrane permeabilization process are not clear, but
it is possible experimentally to observe changes in the macroscopic electric conductance
in membrane model systems due to channel formation. The pores give rise to
leakage of small molecules, such as dithionite or the fluorescent dye calcein, through
the membrane. Models for the pore formation and structure have been much discussed.
33,47-53 In principle, two models are proposed: the “barrel-stave” and the
“torus.” The pore structures can be considered as formed by a bundle of membraneinteracting
peptides with their polar surfaces turned towards the interior of the pore.
With the “carpet” model, the peptide lands on the bilayer leaflet, resulting in a
membrane destabilization, without a defined oligomerization. As mentioned earlier,
the structural modifications of the membrane are considered dynamic on variable
timescales. However, in the case of magainin, a pore structure has even been crystallized
and characterized by neutron diffraction. 53 For most pore-forming peptides
there appears to be a good correlation among pore formation measured as leakage
of the calcein dye, translocation of the peptide through the membrane, and the flipflop
process of membrane lipids.
The peptide translocation in vesicles was measured by observing the fluorescence
resonance energy transfer between a tryptophan residue of the peptide and a
fluorophore (acceptor) attached to the head group of a phospholipid. With a proper
artificial fluorophore bound to the peptide, its translocation in a vesicle can also be
followed by determination of accessibility of the peptide on the outside.