Letter
Figure 3. Normalized fluorescence intensity as a function of time after
addition of a solution of DOPC vesicles loaded with 10 mol % relative
to the lipid (red), 1 mol % (blue), and 0.1 mol % (green) 1 to a 0.1
μM solution of α-synuclein fibrils in PBS 1× (the bulk concentration
of 1 was 15 nM in all cases). Excitation at 440 nm and emission at 490
nm.
Equilibrium was established after a period of 2 h. The rate of
equilibration was independent of the amount of vesicle
vesicles (see Figure S6). Figure 3 shows that the rate of
equilibration increases with the loading of 1, i.e. the local
concentration of 1 in individual vesicles. These observations
suggest that the slow process shown in Figure 3 is not
equilibration of protein−dye interactions between different
vesicles; rather, it is equilibration of protein−dye interactions
Figure 5. (a) Fluorescence spectra for titration of DOPC vesicles
containing 10 mol % 1 into 0.1 μM α-synuclein fibrils in PBS 1× (λex
= 440 nm). The initial spectrum is highlighted in red, and the final
point of the titration in black. (b) Fluorescence emission intensity at
λem = 490 nm for titration of DOPC vesicles containing 10 mol % 1
into 0.1 μM α-synuclein fibrils (squares) and into PBS buffer (circles).
The lines are the best fit to a model that accounts for the emission
due to the complex between the dye and the protein and for the
emission due to the free dye.
17
on the surface of individual vesicles.
It is possible that the slow process is due to further
aggregation of the α-synuclein fibrils. However, transmission
electron microscopy of protein samples exposed to DOPC·1
vesicles showed no evidence for the formation of larger fibers.
fluorescence emission of the free dye as well as the
fluorescence emission of the complex formed when the dye
is bound to the α-synuclein fibrils. The dissociation constants
for the interaction with α-synuclein fibrils are summarized in
Table 1. There is an increase of two orders of magnitude in
Table 1. Dissociation Constants (K ) and Fluorescence
d
Amplification Factors (Ibound/Ifree) for Interaction with 0.1
μM α-syn Fibrils at pH 7.4 and 298 K Measured Using
a
Fluorescence Titrations
b
compound
lipid
Kd (nM)
Ibound/Ifree
ThT
1
830 ± 80
70 ± 20
15 ± 1
4 ± 1
23 ± 13
370 ± 260
91 ± 43
10 ± 2
Figure 4. Transmission electron microscopy images of (a) sonicated
α-synuclein fibrils, (b) fully aggregated α-synuclein fibers, and (c)
sonicated α-synuclein fibrils incubated for 3 h with 150 nM DOPC
vesicles loaded with 10 mol % 1 in PBS buffer.
1
1
(10 mol %)
(1 mol %)
DOPC
DOPC
a
Average values from repeated experiments with standard deviations.
Loading of 1 expressed as molar percentage relative to lipid.
b
incubated with DOPC·1 vesicles for 3 h are very similar to the
starting α-synuclein fibrils (Figure 4a) and quite different from
a sample of fully aggregated α-synuclein fibers (Figure 4b).
The binding affinity for α-synuclein fibrils was measured by
adding increasing amounts of DOPC·1 vesicles and allowing
the solutions to equilibrate for 3 h before recording the
fluorescence intensity. Figure 5 shows the results of a typical
fluorescence titration experiment and the best fit to a 1:1
binding isotherm based on the concentration of 1. Figure 5b
compares the fluorescence titration data obtained in the
presence and absence of α-synuclein fibrils. The DOPC·1
vesicles have a low fluorescence emission intensity in the
absence of protein, and there is an enhancement of more than
an order of magnitude in the fluorescence emission intensity
on binding to the fibrils.
binding affinity for DOPC·1 vesicles compared with the parent
15
dye, ThT. Compound 1 also binds α-synuclein fibrils with a
reasonably high affinity in the absence of vesicles, which
suggests that there are additional binding interactions between
hydrophobic regions on the surface of the fibril and the lipid
tail. In this case, there was no time-dependent change in the
fluorescence intensity, so equilibration of the samples was not
required. This observation is consistent with the hypothesis
that reorganization of the dyes at the lipid membrane interface
is responsible for the slow process shown in Figure 3.
The increase in fluorescence emission intensity associated
with binding of the dye to the protein was quantified using the
fluorescence amplification factor (Ibound/Ifree). The amplifica-
tion factor was calculated as the ratio of the fluorescence
emission due to bound and free dye in a solution, which
contains free dye at a concentration equal to the dissociation
The titration data for addition of the dye to the protein
solution and the dilution data for addition of the dye to buffer
were simultaneously fit to an isotherm that accounted for the
6
48
Org. Lett. 2021, 23, 647−650