Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 295
Figure 8.25. Quenching of tryptophan residues in a membrane-spanning
peptide by a hydrophobic nitroxide. Revised and reprinted with
permission from [61]. Copyright © 2003, American Chemical
Society.
Figure 8.26. Comparison of acrylamide and nitroxide quenching of
tryptophan residues in a membrane-spanning peptide. Revised and
reprinted with permission from [61]. Copyright © 2003, American
Chemical Society.
on the position of the trp residue relative to the center of the
bilayer. The most accessible trp residue is located 9 positions
from the center, and is the most protected residue in
the center of the bilayer.
These membrane-bound peptides were also studied
with hydrophobic quencher 10-doxylnonadecane (10-DN).
This quencher (19 carbons in total length) should be able to
bind to the DOPC bilayer without folding back on itself.
The alkyl chains on each side of the doxyl group are equivalent
in length. Hence the doxyl group was expected to
localize at the center of the bilayer. Figure 8.25 shows
quenching of the trp peptides in DOPC bilayers by 10-DN.
10-DN is not significantly soluble in water and the Stern-
Volmer plot is presented in terms of the mole% of 10-DN in
the bilayers. The Stern-Volmer plots curve upward but it is
still clear that the trp residue at the center of the bilayer is
most strongly quenched, and the extent of quenching by 10-
DN decreases as the trp residue is displaced from the center
of the bilayer. The effect of trp position on quenching is
summarized in Figure 8.26. The extent of quenching by
acrylamide, which is water soluble, decreases as the trp
residue is displaced from the center of the bilayer. The
extent of trp quenching by 10-DN shows the opposite
dependence. Such experiments can be used to determine the
location of tryptophan or other fluorophores in membranes
when the structures are not known.
8.10.3. Quenching of Membrane Probes Using
Localized Quenchers
The previous example showed that accessibility of a fluorophore
to a quencher can be used to roughly indicate its
position in a membrane. However, this approach did not
provide an estimate of its actual location. A somewhat more
advanced approach is to use quenchers that are covalently
linked to the phospholipids and thus restricted to particular
depths in the lipid bilayers. Typical lipid quenchers are the
brominated phosphatidylcholines (bromo-PCs) and nitroxide-labeled
PCs or fatty acids (Figure 8.27). This approach
was used to determine the locations of the fluorenyl groups
in a series of fluorenyl fatty acids when bound to erythrocyte
ghosts (Figure 8.28). The term "ghosts" refers to the
red-blood-cell membranes following removal of hemoglobin
by cell lysis. The fluorenyl probes were quenched by
9,10-dibromostearate that partitioned into the membrane. 62
Since the bromine atoms are rather small, they are expected
to be localized according to their position on the fattyacid
chain. The bromine atoms are located near the center