Diphtheria Toxin NR and SPR
Measurements
Rebecca Eells, Frank Heinrich, Mykola Rodnin, Mathias Lösche, Alexey
Ladokhin
December 2013
1
2
Summary………………………………………………………………………..1
SPR………………………………………………………………………………2
2.1 December 2013 – Peripheral Form…………………………………………...2
2.2 January 2014 - Fully Inserted Form….……………………………………….3
2.3 Data Fitting……………………………………………………………………..5
3
NR……………………………………………………………………………….7
3.1 June 2013 - Fully Inserted Form.……………………………………………..7
3.1.1
Experimental …………………………………………………………...7
3.1.2
Results…………………………………………………………………..8
3.2 November 2013 - Peripheral Form…………………………………………..11
3.2.1
Experimental…………………………………………………………..11
3.2.2
Results………………………………………………………………….12
3.3 Comparison……………………………………………………………………15
3.4 Logbooks……………………………………………………………………….17
3.4.1
June 2013 – Fully Inserted Form……………………………………..17
3.4.2
November 2013 – Peripheral Form………………………………….19
1 Summary
1
2 SPR
2.1 December 2013 – Peripheral form
Two SPR experiments (see Figure 1 and Figure 2) were conducted at conditions
associated with a peripheral binding mode of DTT to the lipid bilayer: 90:10
POPC:POPG, pH 5.5. Both experiments are consistent. At concentrations below
250 nM there is little protein association and a net decrease in the SPR signal by
about 1-3 pixels. This decrease might be due to membrane thinning, as it is
observed by NR under those conditions. Concentrations of 250 nM and 500 nM
show a reversal to a positive net change of the signal by less than 5 pixels. This is
consistent with the NR experiment at 500 nM, in which only a small surface
density of protein was observed. When incubating the lipid bilayer with 1µM, a
steady increase of the SPR signal is observed without entering an equilibrium
state, even after 2h. This signature is consistent with protein aggregation. NR
experiments at 2.5 µM did not show any membrane-associated protein, which
might be a result of aggregation, as well. The bilayer is located at the bottom of
the SPR cell, such that large aggregates would be falling onto the interface,
whereas in the neutron cell the membrane is in a vertical orientation.
90:10 POPC:POPG, pH 5.5
position SPR minimum / pixels
465
as-prepared bilayer
10 nM
20 nM
50 nM
100 nM
250 nM
500 nM
1 µM
460
455
450
445
440
0
50
100
150
time / min
2
200
250
bilayer
c1
c2
c3
c4
c5#1
c6
c7#2
Figure 1: SPR response (proportional to mass at the interface) upon incubation of a 90:10 POPC:POPG
tethered bilayer with DTT. (15-Dec-2013)
90:10 POPC:POPG, pH 5.5
position SPR minimum / pixels
465
as-prepared bilayer
10 nM
20 nM
50 nM
100 nM
250 nM
500 nM
1 µM
rinse
460
455
450
445
0
20
40
60
80
time / min
100
120
140
Figure 2: SPR response incubation of a 90:10 POPC:POPG tethered bilayer with DTT.
Vertical shoot-ups are most likely instrument-related.
2.2 January 2014 – Fully Inserted Form
bilayer
c1
c2
(16-Dec-2013).
c3
c4
c5#1
c6
c7#2
c8#2
Two SPR experiments (see Figure 3 and Figure 4) were conducted at conditions
associated with the fully inserted state of DDT into the lipid bilayer: 70:30
POPC:POPG, pH 5.5. The two experiments, however, yielded different results.
For both experiments, there is little to no protein association for concentrations
below 20nM. At 50nM there is a decrease in the SPR signal by about 1-2 pixels,
which is more obvious for the experiment conducted on January 28 (Figure 3).
The two experiments then begin to diverage at the 100nM concentration. For
Figure 3, the 100nM concentration shows a reversal to a positive change of the
signal by about 1-2 pixels, while in Figure 4 the signal shows essentially no
change from the previous concentration. When the lipid bilayer was incubated
with 250nM on January 28 (Figure 3), a steady increase of the SPR signal was
observed without entering an equilibrium state, even after 1 hour. When the
lipid bilayer was incubated with 250nM on January 29 (Figure 4), however, a
positive change in SPR signal by about 1-2 pixels was observed and equilibrium
was reached after approximately 8 minutes. A concentration of 500nM was then
3
added to the system, which resulted in a positive change in SPR signal by about
5-6 pixels and reached equilibrium after approximately 30 minutes. Incubation
with 750nM, however, resulted in a steady increase of the SPR signal was
observed without entering an equilibrium state.
Figure 3: SPR response (proportional to mass at the interface) upon incubation of a 70:30 POPC:POPG
tethered bilayer with DTT. (28-Jan-2014)
4
Figure 4: SPR response (proportional to mass at the interface) upon incubation of a 70:30 POPC:POPG
tethered bilayer with DTT. (28-Jan-2014)
2.3 Data Fitting
A standard SPR binding kinetics curve shows an increase in the SPR resonance
angle (in pixels) as a function of time. Once the curve plateaus (reaches a
constant SPR resonance angle), equilibrium has been achieved and the
measurement is stopped. These standard SPR binding curves can then be fit
using the equation:

(konc0 koff )t
R  R0 1 e

Equation 1
derived from a 1:1 Langmuir bimolecular model of an analyte molecule (protein)
binding to a single ligand molecule (lipid) where R is the SPR response, R 0 is the
response at dissociation time zero, and c0 is the concentration of protein added to
the system.

During the DTT SPR experiments, however, “nonstandard” binding curves were
observed for both peripheral and transmembrane binding conditions. These
nonstandard binding curves resulted in an increase in the SPR signal that did not
reach equilibrium even after 2hrs. The concentration at which the nonstandard
binding curve was observed varied. To begin to understand these nonstandard
binding curves, the curves were first compiled into one graph. The offset for
each curve was then adjusted in order to obtain an overlay of all the curves
5
(Figure 5). Based on the overlay it appears that the behavior of the nonstandard
curves is the same for every experiment.
Figure 5: Overlay of nonstandard SPR binding curves observed after incubation with DTT
A simplified version of equation derived from the Langmuir model of binding
was used to fit the SPR binding curve from the concentration that results in a
steady increase in SPR signal and the curve from the concentration directly
preceding it. In the simplified equation the concentration and on/off rates are
grouped into one coefficient. For the experiments conducted under peripheral
binding conditions (90:10 POPC:POPG) the concentration directly preceding the
nonstandard response was 0.5uM DTT. The SPR response curve and the fit for
the binding of 0.5uM DTT to 90:10 POPC:POPG on December 15, 2013 is shown
in Figure 6. The SPR curve for the binding of 0.5uM DTT can be described by a
single exponential equation consistent with 1:1 Langmuir binding. The SPR
response curve and the fit for the binding of 1.0uM DTT to 90:10 POPC:POPG on
December 15, 2013 is shown in Figure 7. The SPR curve for the binding of 1.0uM
DDT can be described by two exponential equations (with differing coefficients)
connected by a transition region.
6
Figure 6: Fit of the SPR response after incubation of 90:10 POPC:POPG with 0.5uM DTT based on the 1:1
Langmuir binding model
7
Figure 7: Fit of the SPR response after incubation of 90:10 POPC:POPG with 1.0uM DTT based on the 1:1
Langmuir binding model
3 NR
3.1 June 2013 – Fully Inserted Form
3.1.1 Experimental
The lipid bilayer was prepared by rapid solvent exchange. All buffers used are
derived from a 50 mM phosphate buffer pH 8.0. Buffers with lower pH were
prepared by adding acetic acid / acetate buffer, prepared by Mykola. Wild type
DTT was from a 41 µM stock at pH 8.0 and added to the measurement buffer
right before the NR measurement. The protein was incubated for 30 min and
rinsed thereafter. The measurement started after the rinsing.
Filename
al001-02
8
Condition
Neat
bilayer,
Contrasts
50:50 D2O, H2O
al003-04
al005-06
al007-08
al009
βMe:HC18,
70:30
POPC:POPG, pH 5.3
Incubation with 100nM
DTT at pH 5.3 for 30 min,
rinse and measurement
Incubation with 500nM
DTT at pH 5.3 for 30 min,
rinse and measurement
Rinse with buffer at pH
4.5 and measurement
Rinse with buffer at pH
8.0
H2O, D2O
H2O, D2O
D2O, H2O
H2O
3.1.2 Results
DTT fully inserts into the membrane at all measured concentrations, although
the total amount of associated protein is small for the measurement after
incubating with 100 nM. The protein remains at the interface with no significant
reduction in total amount after a rinse of the system at pH 4.5. The protein
envelope spans the entire hydrocarbon chains and the outer lipid headgroups. A
fit is currently in progress exploring, whether protein is present also in the inner
lipid headgroups. During the current fit the protein envelope was constraint not
to penetrate this region. In all measurements a low protein density is present that
extends at least 60Å into the bulk solvent.
Table 1: Median fit values and 68% confidence limits. Thicknesses are given in Å, nSLD values are given
in 10-6Å-2, and the amount of protein is given as a volume surface density in Å3/Å2.
Parameter
Substrate
SiOx thickness
SiOx nSLD
Cr thickness
Cr nSLD
Au thickness
Au nSLD
9
100 nM DTT 500 nM DTT Rinse, pH 4.5
incubation,
incubation,
pH 5.3
pH 5.3
11.5±2.0
3.63±0.11
29.0±1.5
3.04±0.02
115.2±0.3
4.54±0.02
12.0±1.8
3.60±0.10
28.7±1.2
3.04±0.02
115.1±0.3
4.54±0.02
14.7±1.8
3.48±0.07
26.7±1.5
3.04±0.02
115.2±0.3
4.53±0.02
Rinse, pH 8.0
Substrate
Roughness
Lipid Bilayer
Molar fraction of
tether in inner lipid
leaflet
Number of bME
molecules
per
tether
Tether thickness
Inner hydrocarbon
thickness
Outer hydrocarbon
thickness
Protein
Thickness change of
hydrocarbons
Amount
of
associated protein
Fraction of protein
retained in second
contrast
Global
χ2
10
2.3±0.6
1.9±0.6
2.3±0.6
0.41±0.16
0.41±0.12
0.43±0.14
3.4±0.9
3.5±0.9
3.5±0.8
10.4±0.2
15.2±0.4
10.9±0.2
16.4±0.5
11.1±0.2
16.1±0.5
13.2±0.4
13.6±0.5
13.8±0.5
+0.5±0.1
+1.2±0.1
+1.2±0.2
1.3±0.5
5.6±1.2
5.3±1.3
0.25±0.60
0.47±0.22
0.83±0.26
1.47
Figure 8: Molecular Distributions of bilayer and protein for 100 nM DTT, pH 5.3 @ 50:50 POPC:PG
Figure 9: Molecular Distributions of bilayer and protein for 500 nM DTT, pH 5.3 @ 50:50 POPC:PG
11
Figure 10: Molecular Distributions of bilayer and protein for the rinsing step at pH 4.5 after the
incubation with 500 nM DTT @ 50:50 POPC:PG. This fit is not yet fully optimized as it is the case for the
500 nM incubation @ pH 5.3, the protein penetration is not allowed to go below the 30Å mark.
3.2 November 2013 – Peripheral Form
3.2.1 Experimental
The lipid bilayer was prepared by vesicle fusion. All buffers used are derived
from a 50 mM phosphate buffer pH 8.0. Buffers with lower pH were prepared by
adding 0.1 M acetic acid / 0.1 M acetate buffer, pH 4.0. Wild type DTT was from
a 41 µM stock at pH 8.0 and added to the measurement buffer right before the
NR measurement. For each concentration the system was first measured, while
the protein was incubating (2 x 6h), and a second time measured after rinsing (2 x
6h). This procedure was chosen over that from June 2013, because it was
expected that the protein under peripheral binding conditions might readily
dissociate from the bilayer after rinse.
Filename
al010-11
al012-13
12
Condition
Contrasts
Neat
bilayer,
70:30 D2O, H2O
βMe:HC18,
90:10
POPC:POPG, pH 5.6
Incubation with 500nM H2O, D2O
al014-15
al016-17
al018-19
DTT
at
pH
5.6,
measurement
during
incubation
Rinse, pH 5.6
D2O, H2O
Incubation with 2500nM H2O, D2O
DTT
at
pH
5.8,
measurement
during
incubation
Rinse, pH 5.8
D2O, H2O
3.2.1 Results
Bilayer-associated protein was only observed during the measurement while
incubation with 500 nM DTT. The protein was mostly dissociated during the
measurement that was taking after rinsing the protein. Surprisingly, reincubating the membrane with 2.5 µM DTT did not restore any protein at the
membrane.
The protein envelope measured during the 500 nM DTT incubation spans the
outer leaflet hydrocarbon chains and headgroups, exhibiting a maximum close to
the hydrocarbon-headgroup interface. The total amount of associated protein is
relatively low with 2.7 ± 1.3 Å3/Å2. As during the June 2013 measurements of the
fully inserted form, there is at least 60Å of extra, low-density material present at
the interface.
Table 2: Median fit values and 68% confidence limits. Thicknesses are given in Å, nSLD values are given
in 10-6Å-2, and the amount of protein is given as a volume surface density in Å3/Å2.
Parameter
Substrate
SiOx thickness
SiOx nSLD
Cr thickness
Cr nSLD
Au thickness
Au nSLD
Substrate
13
500 nM DTT Rinse,
incubation,
5.6
pH 5.6
15.9±1.9
3.42±0.05
34.5±1.9
3.05±0.02
161.5±0.4
4.51±0.02
4.1±0.6
16.3±1.8
3.42±0.04
34.0±1.9
3.05±0.02
161.5±0.5
4.50±0.02
4.4±0.5
pH 2500 nM DTT Rinse, pH 5.8
incubation,
pH 5.8
15.6±1.8
3.42±0.05
34.7±1.7
3.05±0.02
161.6 ±0.4
4.49±0.02
4.0±0.5
15.8±1.8
3.41±0.05
34.6±1.9
3.05±0.03
161.7 ±0.5
4.53±0.02
4.1±0.4
Roughness
Lipid Bilayer
Molar fraction of
tether in inner lipid
leaflet
Number of bME
molecules
per
tether
Tether thickness
Inner hydrocarbon
thickness
Outer hydrocarbon
thickness
Protein
Thickness change of
hydrocarbons
Amount
of
associated protein
Fraction of protein
retained in second
contrast
14
0.67±0.20
0.76±0.20
0.67±0.18
0.63±0.19
2.7±1.0
2.5±1.0
2.8±1.0
3.0±0.9
13.4±0.3
16.5±0.7
13.2±0.2
16.4±0.5
13.3±0.2
16.4±0.4
13.3±0.2
16.1±0.5
14.6±0.5
14.6±0.4
14.6±0.4
14.6±0.3
-0.09±0.11
-0.2±0.1
-0.2±0.1
-0.3±0.1
2.7±1.3
1.4±0.7
0.2±1.1
0.3±0.9
0.57±0.60
1.6±1.5
1.4±2.1
1.0±2.3
Figure 11: Molecular Distributions of bilayer and protein for 500 nM DTT, pH 5.6 @ 90:10 POPC:PG
Figure 12: Molecular Distributions of bilayer and protein for the rinsing step after incubation with 500
nM DTT, pH 5.6 @ 90:10 POPC:PG
Figure 13: Molecular Distributions of bilayer and protein for 2500 nM DTT, pH 5.8 @ 90:10 POPC:PG
15
Figure 14: Molecular Distributions of bilayer and protein for rinsing step at pH 5.6 after incubation of
2500 nM DTT @ 90:10 POPC:PG
3.3 Comparison
Figure compares protein envelopes obtained for the 500 nM DTT at conditions
that favor peripheral protein association (90:10 POPC:POPG, pH 5.6) and
conditions for trans-membrane insertion (70:30 POPC:POPG, pH 5.3). At
peripheral conditions only little membrane-associated protein is observed, and
the envelope has been scaled to match the initial slope of the protein envelope
obtained under trans-membrane conditions. Uncertainties are therefore relatively
large for the peripheral protein envelope. There is no significant difference in
membrane penetration for the two conditions. The extension of the extramembranous density is also consistent between the two measurements. The
envelope obtained under trans-membrane conditions shows and increased
protein density in the outer headgroup region.
While structurally, the envelopes are very similar, the association of the protein
under peripheral conditions was reversible, while under trans-membrane
conditions it was not. Protein under trans-membrane conditions was incubated
only for a short time, then rinsed and measured. Under peripheral conditions the
16
envelope was determined while the protein was still in solution. After rinsing the
system, NR did not detect a significant amount of protein, anymore.
Figure 15: Comparison of protein envelopes obtained for the 500 nM DTT at conditions that favor
peripheral protein association (90:10 POPC:POPG, pH 5.6) and conditions for trans-membrane insertion
(70:30 POPC:POPG, pH 5.3).
17
3.4 Logbooks
3.4.1 June 2013 – Fully Inserted Form
18
19
3.4.2 November 2013 – Peripheral Form
20