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170 Cell-Penetrating Peptides: Processes and Applications
maximum emission and the quantum yield. 97 Others, such as polarization and lifetime
measurements, are slightly more sophisticated and require specialized accessory
equipment. 98
The position of the emission spectrum’s maximum provides information on the
Trp environment. A low wavelength maximum (around 330 nm) is indicative of a
Trp in a nonpolar environment, i.e., in contact with the phospholipid hydrocarbon
chains; a high wavelength maximum (around 355 nm) reflects a polar environment
close to the phospholipid headgroups.
Other types of measurements based on fluorescence properties, such as fluorescence
transfer and quenching experiments, can be carried out. The latter deal with
accessibility of quenchers such as I – , Cs + , trichloroethanol, or acrylamide to the
probe, 99 while the former can be carried out using fluorescently labeled lipids.
8.2.2.6 Electron Paramagnetic Resonance (EPR) Spectroscopy
Similarly, to fluorescence, EPR spectroscopy also provides information about the
interaction between peptides and membranes. This spectroscopic method is seldom
used since it requires introducing the presence of side chain reactive residues such
as cysteine in order to introduce an extrinsic spin label. The analysis of EPR spectra
(width of the central line and peak-to-peak heights) provides the rotational correlation
time, which is related to mobility of the particle (the vesicle containing the
peptide) and that of the membrane-embedded peptide. For example, using this
method, it was shown that spin-labeled Cys-pAntp binds DMPG or DOPG vesicles
and not the neutral DMPC vesicles. 49
8.2.2.7 Atomic Force Microscopy (AFM)
AFM allows observation of nanometer-sized particles and provides information on
topographical organization of transferred monolayers 100-104 or bilayers. 105-108 AFM
observation of monolayers requires transfer to a solid substrate and provides details
on the domains existing in phase-separated thin films together with the localization
of the protein. Generally, the observations deal with the hydrophobic side of the
monolayer. Bilayers, which are more biologically relevant, are obtained by double
transfer or by fusion of small unilamellar vesicles to a glass or mica surface;
observations are made on the hydrophilic face of the bilayer. They allow identification
of membrane domain and their constituents.
A final remark about the various methods reported here concerns their joined
use. Indeed, it is highly recommended to describe the behavior of a lipid-interacting
peptide using several approaches; otherwise, some dramatic misinterpretations can
be made. This will be illustrated in an example in the next section.
8.2.2.8 An Example of Multidisciplinary Approach Based on Monolayers
A peptide based on the association of a signal peptide with a sequence issued from
a positively charged nuclear localization motif (SP–NLS) (Table 8.1) was shown to
act as a very efficient oligonucleotide carrier with a very rapid internalization process.
In order to deepen understanding of the membrane-crossing process, a detailed study