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Biophysical Studies of Cell-Penetrating Peptides 235
different paramagnetic probes was not clear-cut for all residues, however, indicating
that the systems are highly dynamic. In experiments with tryptophan fluorescence, the
quenching properties of the two residues in penetratin were indistinguishable. 7
In transportan and penetratin bound to the SDS micelles, the helical parts of the
peptides are composed of 10 to 12 residues making about three turns of a helix
(spanning approximately 16 Å). This corresponds to only about half the width of a
typical phospholipid double layer. The possible importance of these features of the
micelle-bound CPP for the translocation mechanism will be speculated on at the
end of this chapter.
10.6.3 STRUCTURE INDUCTION IN BICELLES
Another recent approach is to use bicelles, disk-shaped objects composed of a
suitable mixture of phospholipids with longer and shorter fatty acid chains. The
phospholipds with the longer chains (e.g., DMPC) assemble to a bilayer making up
the flat part of the disk, and the phospholipids with the shorter chains make up the
rim. Certain preparations with suitable ratios of the phospholipid components give
bicelles with molecular weights down to about 200 kD. Neutral and charged phospholipds
may be mixed to obtain a variable surface charge of the bicelle. When
bicellar solvents are used, it appears that relatively well-resolved NMR signals can
be obtained from the peptide, but few reports on studies in such solvent systems
have been published until now, 24 and not one with a CPP.
10.6.4 STRUCTURE INDUCTION IN VESICLES
As already pointed out, the more realistic membrane-mimetic systems are larger
objects that are not amenable to high-resolution NMR studies. The practical limit
for molecular weights directly accessible by NMR is around 50 kD, extended by
transverse relaxation optimized spectroscopy (TROSY) techniques to a few hundred
kD under favorable circumstances. 37 Certain ways around this practical problem
have been devised, one of which is to use transferred nuclear Overhauser enhancement
(NOE) experiments for structural information on peptides, which must be only
weakly bound to the vesicle and therefore in rapid exchange between the free and
bound states. These experiments combine the sharp linewidth of the peptide free in
solution with some structural information (in terms of NOEs between resonances
from protons close in space) on the bound peptide.
CD spectroscopy has been a method of preference for studies of structure
induction by phospholipid vesicle systems. The overall conclusion for penetratin is
that the charge of the membrane surface determines not only the strength of the
interaction but also the dominating nature of the induced secondary structure. With
highly negatively charged vesicles or high peptide–lipid ratios, the induced secondary
structure is mainly a β-structure, whereas more neutral vesicles or low peptide–lipid
ratios favor an α-structure. 43-45 This chameleon-like behavior of penetratin
seems not to be followed by transportan, however, which keeps its partial α-structure
unchanged, in phospholipid vesicles of different charges as well as in SDS micelles
or in a 30% HFP mixture.