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Biophysical Studies of Cell-Penetrating Peptides 239
Interestingly, it has recently been reported 58 that penetratin could induce a
transient aggregation of LUVs produced from a pure acidic phospholipid (DOPG).
In this study a high peptide/lipid molar ratio was used, which should lower the
surface potential. The aggregation was correlated with the occurrence of a concomitant,
transient β-structure induction in the penetratin peptide. Upon the time-dependent
disappearance of the vesicle (LUV) aggregation, the secondary structure of the
peptide returned to an α-helical state. The authors suggested that peptide translocation
could be responsible for this time-dependent phenomenon: if the penetratin
escapes to the inside of the (aggregated) LUVs, the peptide surface concentration
will become lower, which should favor the helical state and also electrostatically
improve the colloidal stability of the vesicles. In summary, further critical studies
with penetratin and other CPPs are required in order to establish and characterize
any translocation in relevant membrane model systems.
10.9 CRITERIA FOR VARIOUS FORMS OF PEPTIDE ACTIVITIES
ON MEMBRANES
In this context we would like to emphasize that it is important to define simple and
robust criteria for the interpretation of the different terms: pore formation, translocation
with cargo, etc. The meaning of these terms in a biological cell context is
relatively clear; this is how the remarkable properties of the peptides were discovered.
In the membrane model systems, however, there is a need to formulate operational
definitions (protocols) of the different processes. For instance, one may define
pore formation in a phospholipid vesicle by observing leakage above control level
of a certain dye. It should also be emphasized that, although we discuss pore
structures as if they were static, all these phenomena are dynamic and occur on
various time scales. Further, one may define nonmediated translocation through a
phospholipid vesicle membrane from the inside–outside distribution of the peptide.
The distribution (topology) can be observed by fluorescence resonance energy transfer
(FRET) properties between a fluorophore in the peptide and fluorescence-labeled
lipids on either side of the bilayer, or simply by removing or destroying the exterior
peptide fluorophore. Accompanying the peptide translocation, processes may exist
like an induced lipid flip-flop process of the phospholipids across the bilayer.
For obvious reasons it is difficult to establish translocation (permeation) in a
biological cell that undergoes lysis. Translocation in biological systems is therefore
restricted as a phenomenon to such peptides, which do not cause drastic perturbances
of the cell membrane. One could imagine a quantitative rather than qualitative
difference between the pore-forming and translocating groups of peptides; CPPs
may constitute a group with similar but milder effects on membrane integrity than
the antimicrobial ones. In both cases, peptides should concentrate at the membrane
surface. The interaction may cause weakening of the membrane barrier, which, in
the pore forming case, will result finally in lethal permeability.
For good CPPs, on the other hand, disruption of the phospholipid assembly
should be less serious and permanent, perhaps because the peptide does not so easily
form aggregates at the membrane surface, or because it is only partially inserted