crc press - E-Lib FK UWKS

Interactions of Cell-Penetrating Peptides with Membranes 169
8.2.2.3 Lipid-Containing Air–Water Interface
Biological membranes are composed of lipid bilayers that constitute the major barrier
for cell-penetrating peptides. These peptides are involved in exchange processes and
lipid–peptide interactions may modify the lipid packing and thus the membrane
properties. 67-72
Monomolecular films are good investigative models since the thermodynamic
relationship between monolayers and bilayer membranes is direct, and monomolecular
films at the air–water interface overcome limitations such as regulation of lipid
lateral-packing and lipid composition that occur in bilayers. 73 Measurements of
peptide–lipid interactions can be achieved by forming a lipid monomolecular film
at a given surface pressure close to the maximum obtained for the peptide at the
air–water interface, 74 and by injecting aliquots of the peptide solution into the
subphase. 74-77
Mixed lipid–protein films can also be obtained by spreading on the water surface
a mixture of the peptide and lipid dissolved in a volatile solvent 78-80 or by deposition
of vesicles containing the desired mixture. 81 The conformational analysis of peptides
interacting with lipids in an interfacial situation can be made by FTIR on Langmuir–Blodgett
films transferred onto a germanium plate 82,83 or by CD when the
transfer is made onto quartz plates. 84,85 Another advantage of monolayer lies in the
fact that the lateral pressure can be varied and thus allows study of the influence of
this pressure on the conformational state of the peptide. 86 However, such analyses
have to be made with care since the transfer process can induce structural modifications.
Thus, in situ methods have been developed accordingly. 87,88
8.2.2.4 Bilayers
Contrary to monolayers, bilayer properties are more restricted but better models for
plasmic membranes; therefore, most investigations have been carried out with liposomes.
After uptake of the peptide by the liposomes, conformational analysis can
be carried out by CD, x-ray or infrared spectroscopy. 89-93 For helix-rich peptides,
insertion into the bilayers induces pore formation, 94 while other peptides interact
only with receptors that are membrane-embedded. These receptor–peptide interactions
can induce conformational changes and in some cases, can promote fusion
processes. 95 Peptide-embedded analysis can also be carried out on bilayers obtained
by the transfer protocol by Langmuir–Blodgett or Langmuir–Schaeffer methods. 96
8.2.2.5 Fluorescence
Most proteins contain intrinsic fluorophores, which are the aromatic amino acids
tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). Protein fluorescence is
generally obtained by excitation at the absorption maximum near 280 nm or higher
wavelengths. Under these conditions, Phe is not excited and hence cannot be considered
an appropriate probe for fluorescence investigations on proteins. In fact, the
most useful residue is Trp, since it can be selectively excited from 295 up to 305 nm
and is sensitive to both specific and general environment effects. Briefly, two phenomena
are currently used to describe the environment of a Trp residue: the wavelength of the