Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 293
Figure 8.20. Acrylamide Stern-Volmer plot for NATA (x), and three
single-tryptophan mutants of the channel-forming peptide of Colicin
E1 at pH 3.5, W-355 (!), W-460 (), and W-443 (O). Revised and
reprinted with permission from [55]. Copyright © 1993, American
Chemical Society.
Figure 8.21. Emission maxima (top) and bimolecular quenching constants
(bottom) of the twelve single-tryptophan mutants of the channel-forming
peptide of Colicin E1 at pH 3.5. Revised and reprinted
with permission from [55]. Copyright © 1993, American Chemical
Society.
The conformation of the membrane-bound form of the
colicin E1 channel peptide was studied by acrylamide
quenching. 55 Twelve single tryptophan mutants were
formed by site-directed mutagenesis. The tryptophan
residues were mostly conservative replacements, meaning
the trp residues were placed in positions previously containing
phenylalanine or tyrosine. Acrylamide Stern-Volmer
plots of three of these mutant proteins are shown in Figure
8.20. The accessibility to acrylamide quenching is strongly
dependent on the location of the residue, and all residues
are shielded relative to NATA. Depending on position, the
trp residues also showed different emission maxima (Figure
8.21, top). The acrylamide bimolecular quenching constants
were found to closely follow the emission maxima,
with lower values of k q for the shorter-wavelength tryptophans
(bottom). Such data can be used to suggest a folding
pattern for the channel-forming peptide, and to reveal conformational
changes that occur upon pH activation of colicin
E1.
8.10. APPLICATION OF QUENCHING
TO MEMBRANES
8.10.1. Oxygen Diffusion in Membranes
Quenching by oxygen has been used to determine the
apparent diffusion coefficient of oxygen in membranes.
This can be accomplished using probes that partition into
the lipid bilayers or are covalently bound to the lipids. Figure
8.22 shows oxygen Stern-Volmer plots for 2-methylanthracene
in vesicles of DMPC and DPPC, which have
phase-transition temperatures (T c ) near 24 and 37EC,
respectively. 56 At the experimental temperature near 31EC
the DPPC bilayers are below the phase transition, and the
DMPC bilayers are above the phase transition. While
anisotropy measurement on such a bilayer suggests a large
change in viscosity at the transition temperature (Chapter
10), the effect on oxygen diffusion is only modest: near
twofold. This surprisingly small change in the oxygen diffusion
coefficient has been confirmed by other fluorescence
57–59 and ESR experiments. 60 These experiments even
suggest that cholesterol, which generally makes membranes
more rigid, results in increased rates of oxygen transport.
This can be seen by quenching of pyrene dodecanoic acid
(PDA) in erythrocyte ghost membranes (Figure 8.23). In
this case, the ghost membranes were modified by addition
of endogenous cholesterol. The apparent bimolecular
quenching constant increases with increasing amounts of
cholesterol, except for the highest membrane concentration