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crc press - E-Lib FK UWKS

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166 Cell-Penetrating Peptides: Processes and Applications<br />

8.2.1 ANALYTICAL METHODS: CONFORMATIONAL IDENTIFICATIONS<br />

8.2.1.1 NMR<br />

Besides crystal structure resolution, NMR is the most precise method for detailed<br />

identification of proteins and peptides structures. However, some limitations lie in<br />

the molecular weight of the particle under examination. While the structure of a<br />

protein of ~150 residues can be easily solved, an increase of its molecular weight<br />

by oligomerization or by incorporation into a lipidic medium (vesicles, for example)<br />

precludes obtaining resolved spectra. For peptides interacting with membranes, most<br />

investigations were carried out in the presence of micelles considered membranemimicking<br />

media and giving rise to resolved spectra. However, the question of<br />

membrane-relevance of the data obtained in such a medium must be raised. Indeed,<br />

nearly all studies show that peptides engaged in a micelle structure adopt a helical<br />

conformation. For example, this holds true for transportan 46 and for the primary<br />

amphipathic vectors based on the SP or FP sequences associated with the NLS<br />

motif. 47,48<br />

In the presence of phospholipids the situation differs strongly from that described<br />

earlier. While the SP-NLS peptides can adopt either an α-helical or a β-type structure,<br />

depending on the peptide/lipid ratio, no helical form could be detected for the<br />

FP–NLS peptide that remains in a β-sheet form, except 6 in Table 8.2, especially<br />

designed to adopt an α-helical conformation.<br />

This remark does not imply that, when a helical form is found in SDS, the same<br />

form does not exist when the peptide is in the presence of phospholipids. Thus, the<br />

extrapolation of the findings in SDS to phospholipids must be taken very cautiously,<br />

especially when the peptides are nonordered in solution in water. On the contrary,<br />

when peptides show a tendency, even low, to adopt a helical conformation, measurements<br />

in SDS micelles are appropriate for structural identification. 49 This problem<br />

of conformational identification is overcome by using solid-state NMR, which<br />

has a great deal of potential for studies of membrane-embedded peptides. 50,51<br />

There are two fundamentally different types of structural constraints that can be<br />

obtained from solid-state NMR experiments. Distance constraints can be obtained<br />

from rotational resonance experiments. Unlike nuclear Overhauser effect constraints<br />

in solution NMR, distance constraints obtained in solid-state NMR are typically far<br />

more quantitative; the distances observed can be 1 nm or even greater, depending<br />

on the gyromagnetic ratios of the interacting nuclei. Such experiments do not require<br />

one-, two-, or three-dimensional order because the samples are spun in a magic<br />

angle spinner.<br />

A second approach for structural constraints is to obtain orientational constraints<br />

by solid-state NMR. For this approach, samples need to be aligned with respect to<br />

the magnetic field direction, B 0, of the NMR spectrometer. Then the orientationdependent<br />

magnitude of numerous nuclear spin interactions such as the anisotropic<br />

chemical shift and dipolar and quadrupolar interactions can be observed. Therefore,<br />

the quality of oriented samples should be high. Thus, the choice of lipid is also<br />

important from the standpoint of having a match between the lengths of the hydrophobic<br />

regions of the peptide or protein and lipid bilayer. Any peptide not in a lipid

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