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168 Cell-Penetrating Peptides: Processes and Applications
Langmuir trough. A considerable improvement was provided by polarization modulation
of the incident light (PMIRRAS), which has proved to be an efficient way
to greatly increase surface absorption detectivity while getting rid of the intense
isotropic absorption occurring in the sample environment. 61,62
8.2.1.4 Diffraction
Since protein structure determination by classical x-ray crystallography is difficult
to obtain for membrane protein, the alternative is to grow two-dimensional crystals
by adsorbing proteins to ligand–lipid monolayers at the air–water interface. Using
grazing incidence synchrotron x-ray diffraction and a setup of high angular resolution,
narrow Bragg reflections are observed corresponding to a resolution of 10 A
in plane and 14 A normal to the plane in the case of streptavidin. This approach is
complementary to electron diffraction but avoids the process of transfer onto a grid
and has the advantage of allowing in situ investigations. 63
8.2.2 INTERACTIONS WITH PHOSPHOLIPIDS AND THEIR CONSEQUENCES
8.2.2.1 The Monolayer Approach
Monolayers at the air–water interface are appropriate tools for investigating phospholipid–peptide
interactions. Indeed, analysis of the adsorption or compression
isotherms provides information on miscibility of the components forming the monolayer,
together with information on interactions between these components. Analysis
also provides crucial information concerning the state of the monolayer (below or
above the collapse pressure or squeezing out of one monolayer component), when
applying other methods that allow in situ observations such as infrared or grazingincidence
x-ray diffraction (see above). The management of this monolayer approach
first requires a study in the absence of lipid; the influence of the presence of lipid
is investigated in a second step.
8.2.2.2 Lipid-Free Air–Water Interface
Amphipathic peptides are surface-active molecules that, when in solution in water,
adsorb spontaneously at the solution surface, thus lowering the surface tension γ.
This parameter is measured by the Wilhelmy method and its variation as a function
of the peptide concentration in the subphase provides the adsorption isotherm.
Another method for studying proteins at the air–water interface consists in deposition
as a drop of an aqueous solution of the peptide or as a drop of a solution in a volatile
solvent on the water surface. The peptide molecules are organized at the surface in
a monomolecular film that can be compressed using an hydrophobic barrier in order
to increase the surface density. However, it is sometimes difficult to correlate the
adsorption and compression isotherms because of conformational changes of the
peptide during their adsorption process.
In order to identify conformational changes, transfer experiments can be carried
out. These Langmuir–Blodgett transfers 64 are made at constant surface pressure onto
solid supports such as germanium or CaF 2 for IR analyses. 65,66