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238 Cell-Penetrating Peptides: Processes and Applications
There should be a difference in the biological activities between the antimicrobial
(pore-forming) peptides and CPPs, in that the latter must be much less cell toxic by
their nature or design. At the molecular level, a superficial look at the peptide
sequences shown in Tables 10.1 and 10.2 reveals very few obvious differences:
positive charges and hydrophobic residues are well represented in both groups. An
interesting reflection is that a peptide sequence such as mastoparan, which, like
mastoparan X, belongs to the pore-forming group, may convert into the CPP group
by acquiring an additional new N terminus from galanin. It seems as if the addition
of a somewhat less hydrophobic sequence part makes the molecule less toxic and
at the same time confers ability to carry conjugated cargoes across the membrane.
10.8 CPP TRANSLOCATION IN MODEL MEMBRANE SYSTEMS:
THE CASE OF PENETRATIN
Some basic understanding of the mechanisms behind the CPP translocation phenomenon
is urgent and challenging. For that reason, studies on membrane model
systems are usually required. Will any of the CPPs, even without a cargo, be able
to come across a bilayer of a unilamellar phospholipid vesicle? If the answer is yes,
we need not speculate about any still unknown biological factor responsible for the
translocation. We then just need to acquire better understanding of the biophysics
of membranes. The thermodynamic energy dependence of the transport will be
described in either a classical or an irreversible sense.
The first direct observation of translocation of penetratin across a vesicle membrane
was reported by Thorén et al., 56 who used fluorescence microscopy to observe
labeled penetratin in the presence of giant vesicles (GUVs) prepared from soybean
lipids. The authors reported that translocation took place with GUVs and with
multilamellar liposomes (LMV). Even at the high peptide/lipid molar ratio (1:46)
applied, penetratin did not cause any detectable membrane leakage.
In contrast, no evidence for any translocation was found by Drin et al. 57 when
using fluorophore-labeled penetratin exposed to charged SUVs prepared from a
POPC/POPG mixture (75/25) in saline. The peptide/lipid molar ratio was low (1:708)
in this case. The penetratin remained on the outer leaflet of the SUVs, so the
impermeable dithionite reagent could chemically extinguish the fluorescence. Similarly,
no evidence of translocation was found when penetration was exposed to
charged LUVs at a low peptide/lipid molar ratio. 43
What may be the reasons for the conflicting results with the fluorescence methods?
First, different vesicle types were used. GUVs seem to have relevant biomembrane
mimetic properties, but a better defined lipid composition should be used.
Whether the method of GUV preparation caused a transmembrane potential is not
clear. The absence of translocation with SUVs might be a result of uneven bilayer
packing, causing a strain too high for an effective transport. Perhaps more important,
the studies were carried out with about one order difference in the peptide/lipid
molar ratio. The surface potential may also have been different. Penetratin, with its
chameleon-like properties, can adopt different secondary structures, depending on
the experimental conditions (peptide/lipid ratio, surface charge, etc.). 45