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Interactions of Cell-Penetrating Peptides with Membranes 167
environment with a bilayer normal aligned parallel with B 0 will result in less wellaligned
peptide and protein preparations.
The choice of the orienting surface is dictated by a desire for relatively rigid
surfaces that can be arranged and maintained in a sample container with the surface
normally parallel to the magnetic field direction. Thin glass plates are appropriate
for such preparations.
8.2.1.2 Circular Dichroism (CD)
CD is frequently used to identify the secondary structures of proteins or peptides;
however, it has some drastic constraints with regard to the sample. For peptides or
proteins in solution it is strongly recommended to record spectra in the far UV
region, down to 180 nm, to obtain dependable information related to the conformation.
Such a low wavelength precludes the use of most common buffers (hepes,
acetate, etc.) and of salts based on chloride or bromide, for example. A membranemimicking
situation requires the presence of phospholipids which will be organized
forming vesicles. Except for a few synthetic phospholipids such as DOPG, which
leads to transparent vesicles, most phospholipids form vesicles leading to milky
solutions or suspensions with high scattering power. One issue to overcome these
difficulties lies in the use of lysophospholipids or detergents such as SDS that form
nonscattering micelles. However, a cautious interpretation of the data is required
since a micelle has geometrical constrains that do not exist for membranes. 47
However, CD can allow us to determine the orientation of helical sections of
peptides or proteins in membrane. To do this, membranes are prepared in a multilayer
array and CD spectra are measured at normal and oblique incident angles with
respect to the planes of the layers. Taking into account the artifacts due to dielectric
interfaces, linear dichroism, and birefringence, this method allows the detection of
orientation modifications when the hydration state is varied. 52
8.2.1.3 Fourier Transform InfraRed Spectroscopy (FTIR)
This spectroscopic approach appears to be one of the most powerful methods for
the identification of peptide and protein secondary structures and thus for determination
of conformational changes upon variations of the environment. 53-60 Under
appropriate conditions this method also allows the determination of orientation of
the peptide or protein with respect to an interface. In addition, infrared spectroscopy
also provides information on all membrane components. Most data reported so far
were obtained from solution and thus cannot account for the relative orientation of
the bound peptide vs. the phospholipids.
To this end, several models have been constructed. Using polarized attenuated
total reflection infrared spectroscopy, determination of the helical order parameter
of a short α-helical peptide (melittin in that case) was revealed. It was also determined
that the orientation of the helix depends on the hydration of the membrane
preparation. Therefore, measurements of a fully hydrated state appear more appropriate,
although they are carried out on monolayers. The first approach was carried
out in situ using infrared refection (IRRAS) on phospholipid monolayers on a