Fluorescence Technologies for Monitoring Interactions Between Biological Molecules In Vitro - Progress in Molecular Biology and Translational Science - page 136

membrane. Basically, the observed enhancement of intensity in the
fluorescence of tryptophan in the presence of LUVs is usually titrated
with increasing concentration of quenchers. The resulting data are then
analyzed by using the Stern-Volmer equation: 17

F 0 = F ¼ þ K SV ½ ½

1 48

where F 0 and F are the fluorescence intensities in the absence and in the
presence of quencher, respectively, [Q] is the molar concentration of
quencher, and K SV is the Stern-Volmer quenching constant. A strong
decrease of the K SV value in the presence of LUV is indicative of a loss of
accessibility of the tryptophan to the quencher, suggesting thus a deeper
insertion of molecule in the lipid bilayer. 87
By specifically labeling fatty acyl chains or polar heads, quenching as well
as enhancement of fluorescence can provide a more accurate mapping of the
bilayer location of a protein. A large number of different labeled phospho-
lipids have also been developed to enable direct FRET on the surface mem-
brane or inside the bilayer. Although more difficult to use, this approach is
more accurate. For example, brominated phospholipids were engineered by
the addition of bromide at specific positions of the acyl chains of phospho-
lipids: that is, position (6, 7), (9, 10), and (11, 12) of a stearoyl fatty acid
chain. From the hydrophobic tail of the acyl chain to the polar head of phos-
pholipids, the use of distinct positions of bromide enables the mapping of the
insertion of peptide in a lipid bilayer by bromide quenching of the trypto-
phan intrinsic fluorescence. 97,98 Thus the resulting depth-dependent
fluorescence quenching profiles enables comparison between different
membrane-active peptides. 87,98

4.3. Probing membrane interactions with specific probes
Based on the principle of solvatochromism, several different fluorescent pro-
bes were developed for specifically sensing membrane environments and
membrane domains. Indeed, membranes cannot be considered simply as
single phospholipid bilayers with a hydrophobic core and a polar surface.
Involving different types of domains with various physical states, the “fluid
mosaic model” of the structure of cell membranes is clearly in agreement
with the dynamics of membrane components throughout the phospholipid
bilayer. 99 Like the physical state of phospholipids, the polarity of the lipid
bilayer, the membrane potential, and the hydration of membrane are param-
eters that contribute to the integrity of the membrane. Studies of protein/