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166 Cell-Penetrating Peptides: Processes and Applications
8.2.1 ANALYTICAL METHODS: CONFORMATIONAL IDENTIFICATIONS
8.2.1.1 NMR
Besides crystal structure resolution, NMR is the most precise method for detailed
identification of proteins and peptides structures. However, some limitations lie in
the molecular weight of the particle under examination. While the structure of a
protein of ~150 residues can be easily solved, an increase of its molecular weight
by oligomerization or by incorporation into a lipidic medium (vesicles, for example)
precludes obtaining resolved spectra. For peptides interacting with membranes, most
investigations were carried out in the presence of micelles considered membranemimicking
media and giving rise to resolved spectra. However, the question of
membrane-relevance of the data obtained in such a medium must be raised. Indeed,
nearly all studies show that peptides engaged in a micelle structure adopt a helical
conformation. For example, this holds true for transportan 46 and for the primary
amphipathic vectors based on the SP or FP sequences associated with the NLS
motif. 47,48
In the presence of phospholipids the situation differs strongly from that described
earlier. While the SP-NLS peptides can adopt either an α-helical or a β-type structure,
depending on the peptide/lipid ratio, no helical form could be detected for the
FP–NLS peptide that remains in a β-sheet form, except 6 in Table 8.2, especially
designed to adopt an α-helical conformation.
This remark does not imply that, when a helical form is found in SDS, the same
form does not exist when the peptide is in the presence of phospholipids. Thus, the
extrapolation of the findings in SDS to phospholipids must be taken very cautiously,
especially when the peptides are nonordered in solution in water. On the contrary,
when peptides show a tendency, even low, to adopt a helical conformation, measurements
in SDS micelles are appropriate for structural identification. 49 This problem
of conformational identification is overcome by using solid-state NMR, which
has a great deal of potential for studies of membrane-embedded peptides. 50,51
There are two fundamentally different types of structural constraints that can be
obtained from solid-state NMR experiments. Distance constraints can be obtained
from rotational resonance experiments. Unlike nuclear Overhauser effect constraints
in solution NMR, distance constraints obtained in solid-state NMR are typically far
more quantitative; the distances observed can be 1 nm or even greater, depending
on the gyromagnetic ratios of the interacting nuclei. Such experiments do not require
one-, two-, or three-dimensional order because the samples are spun in a magic
angle spinner.
A second approach for structural constraints is to obtain orientational constraints
by solid-state NMR. For this approach, samples need to be aligned with respect to
the magnetic field direction, B 0, of the NMR spectrometer. Then the orientationdependent
magnitude of numerous nuclear spin interactions such as the anisotropic
chemical shift and dipolar and quadrupolar interactions can be observed. Therefore,
the quality of oriented samples should be high. Thus, the choice of lipid is also
important from the standpoint of having a match between the lengths of the hydrophobic
regions of the peptide or protein and lipid bilayer. Any peptide not in a lipid