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Biophysical Studies of Cell-Penetrating Peptides 231
intrinsic binding at the membrane is usually described by an adsorption (mass action)
isotherm (e.g., Langmuir) reflecting how the bound molecule (peptide) will become
positioned at a site, until the surface becomes fully occupied (saturated).
10.5 BIOPHYSICAL METHODS TO STUDY
PEPTIDE–MEMBRANE INTERACTIONS
Biomembranes are amphiphilic molecular assemblies able to stabilize peptide conformations
upon interaction. When free, short peptides, as distinguished from globular
proteins, do not normally have a more hydrophilic “outside” and a more hydrophobic
“inside.” Depending on the nature of the residues involved, a peptide will
expose both hydrophobic and hydrophilic side chains to the surrounding medium.
Peptides with low solubility may self-aggregate and form oligomers. Various spectroscopic
methods are used to study the interactions between a peptide and a mixed
solvent.
Among problems studied are, e.g., distribution of the peptide between the aqueous
and amphiphilic phase, location and mobility of the peptide relative to the surface
of the membrane mimetic phase, and induction of secondary structure in the peptide
by the solvent. These studies are relevant for various other classes of peptides, such
as hormones and antibiotic peptides. Other studies concern changes of the membrane
induced by the peptide. Such changes range from complete disassembly (lysis) to
pore formation or more subtle effects, which may be relevant for the group of
peptides under consideration here. The most used spectroscopic techniques are
briefly summarized below.
10.5.1 FLUORESCENCE
The intrinsic fluorescence of tryptophan (or possibly tyrosine) residue may be investigated
in order to determine the polarity of its environment. 29 The tryptophan
emission peak wavelength is sensitive to polarity and shifts from ca. 356 nm in an
aqueous phase to about 320 nm in a hydrophobic phase. In parallel, the quantum
yield becomes enhanced in the hydrophobic phase. Artificial fluorescence probes
can be attached to the peptide, cargo, or membrane and may report on interactions
or change of state of its host. As a cargo, the green fluorescence protein (GFP) may
be used as a natural test molecule. The topology of the peptide and membrane system
may be monitored by experiments using quenchers or detecting fluorescence resonance
energy transfer (FRET) between two fluorophores (donor–acceptor).
Polarization of fluorescence can be used to observe order and dynamics (“fluidity”)
of the membrane and perturbations caused by the peptide. Relatively low
concentrations and volumes are needed for spectroscopic fluorescence studies: typically
a few hundred µl of 1 µ M fluorophore, i.e., about 1 nmol, is enough to give
a good fluorescence signal from tryptophan, as well as from any suitable external
fluorescence probe. With fluorescence microscopy, in principle, single fluorescent
molecules may be monitored by advanced techniques. 31