Membrane Crossover by Cell-Penetrating Peptides: Kinetics and Mechanisms – From Model to Cell Membrane Perturbation by Permeant Peptides - Intracellular Delivery: Fundamentals and Applications - page 181

2.1 Necessity for a Peptide Secondary Structure

for Internalization?

It is clear that peptide - membrane interactions must be of fundamental importance
for the internalization process. Therefore, whether the secondary structure of these
peptides when interacting with membrane, plays a key role in the internalization
properties has been widely studied. Moreover different peptide secondary struc-
tures have been reported for the same peptide. This is certainly a result of the dif-
ferent experimental conditions used in the studies regarding: peptide/lipid (P/L)
ratios and concentrations used, buffer composition (e.g., ionic strength), tempera-
ture at which the experiments were performed as well as the method used to deter-
mine its structure.
Penetratin, the cell-penetrating sequence derived from the third helix of the
Antennapedia homeodomain protein, has been shown to have a strong propensity for
a-helix formation in lipid environments, which suggested first that the helical struc-
ture was necessary for internalization of the peptide (Magzoub et al. 2002 ; Letoha
et al. 2003 ; Lindberg et al. 2003 ; Christiaens et al. 2004 ; Caesar et al. 2006 ; Clayton
et al. 2006 ). But, a recent computational study on the molecular structure of penetra-
tin, in interaction with lipid bilayers, and experiments with various phospholipids
mixtures, have indicated a high structural polymorphism of penetratin (Polyansky
et al. 2009 ). Penetratin could indeed adopt a a-helical, a b-strand or a b-turn confor-
mation depending on the model membrane composition (Magzoub et al. 2002 ;
Clayton et al. 2006 ; Su et al. 2008 ). In addition, it has been underlined that the
a-helical conformation was not mandatory and could even be detrimental to the
membrane translocation properties of penetratin (Derossi et al. 1996 ; Christiaens
et al. 2004 ). Finally a recent study in living cells with high concentrations
(25-50 mM) of penetratin has shown that the secondary structure of the peptide
was found to be mainly random coil and beta-strand in the cytoplasm, and possibly
assembling as beta-sheets in the nucleus (Ye et al. 2010 ). Ye and collaborators report
no evidence of a-helical structure formation by penetratin, although it is possible
(because of limitations with the signal intensity and the lateral spatial resolution
(~0.5 mm) of the Raman microscopy methods) that the peptide could form a-helical
or other transient conformations as it crosses the cell membrane (Ye et al. 2010 ).
Thus, whether a correlation exists between the capacity of a CPP to adopt a
specific structure and its membrane translocation ability, is still a matter of debate.
A recent study with ten different CPPs attempts to classify the peptides in three sub-
groups depending on their physicochemical properties (the secondary structure being
one of them) and correlates those with different internalization pathways (Eiríksdóttir
et al. 2010a, b ). It has been suggested that the structural polymorphism and malleability
of CPPs could be important for the membrane interaction and internalization route
(Deshayes et al. 2008 ). An aspect that has been briefly evocated in the literature is the
relevance of CPP self-assembly in the uptake mechanism. It follows that certain CPPs
(penetratin, transportan, Pep-1, MAPs) can self-assemble, suggesting that they can be
internalized as monomers or aggregates (Pujals et al. 2006 ).