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Biophysical Studies of Cell-Penetrating Peptides 233
limits the applicability to molecular weights less than about 50 kD with conventional
methods.
New methodology developments may raise this limit to a few hundred kD, 37 and
solid-state NMR is applicable with larger objects, but the information content is
more limited. At the present time this means that biomembrane mimetic solvents
that allow detailed determination of atomic structure of an interacting peptide are
limited to mixed organic solvents, detergent micelles, and, possibly, phospholipid
bicelles; small unilamellar phospholipid vesicles and larger objects are not useful
as solvents. NMR spectroscopy requires rather large amounts of sample for detailed
studies, typically 0.5 ml of a 1-mM peptide, i.e., 500 nmol.
Besides studies of the peptide itself, NMR can also be applied to study of the
phospholipids of model membranes. 31 P NMR (and 2 H NMR) has been used extensively
to determine properties of membrane phases such as size, disruption into
extended bilayers, phase transitions, or polymorphism.
10.5.5 ELECTRON PARAMAGNETIC RESONANCE
Electron spin (or paramagnetic) resonance (EPR) spectroscopy can be used to study
CPPs if a spin labeling procedure is used. Spin labels, stable nitroxyl free radicals,
can be attached to the peptide or included among the membrane constituents. 38 The
aim of such studies is to determine interactions and mobility of the spin-labeled
peptide exposed to solvent-containing membranes. The degree of binding and
dynamics of a labeled peptide to a membrane particle can be estimated from EPR
spectra. Information about peptide–peptide interactions may also be obtained. As
with fluorescence labels, it is in principle possible to put one label on the peptide
and another on the membrane and then investigate details of peptide–membrane
interactions. The amount of sample needed for EPR-spin label studies is typically
50 µl of a 10-µ M solution, i.e., 0.5 nmol, which is similar to requirements for
fluorescence and much less than those for NMR.
10.6 EXAMPLES OF SECONDARY STRUCTURE INDUCTION
IN CPPS BY MEMBRANE-MIMETIC SOLVENTS
Many studies have reported induction of secondary structure in CPPs by interaction
with membrane-mimetic solvents. We will not review all, but will give a few typical
examples involving detergent micelles and phospholipid vesicles and illustrating use
of different experimental techniques in the different model systems.
Most short peptides have only weak tendencies for ordered secondary structures
in water; evidence from CD as well as NMR is typically interpreted as originating
from a dominating “random coil” secondary structure. This is also true for CPPs
like penetratin and transportan. Based on CD and NMR studies with aqueous fluoroalcohol
(HFP or TFE) mixtures, an α-helical structure has been reported for
penetratin, 6 and for a chimera of signal peptide sequences with a nuclear localization
sequence (NLS; Table 10.1). 12,13 This situation is typical for many peptides in fluoroalcohol–water
solvents, not just CPPs.