PlyC-B tBLM Experiments
Marilia Barros, Frank Heinrich, Mathias Lösche, Daniel Nelson
November 2013
1 Summary and Discussion ................................................................................ 1
2 The PlyC-B protein .......................................................................................... 2
3 SPR Experiments ............................................................................................ 2
3.1 May 2013 .................................................................................................. 3
3.2 January 2014 ............................................................................................ 5
3.2.1 Non-dialyzed PlyC-B .......................................................................... 5
3.2.2 Dialyzed PlyC-B ................................................................................. 8
4 Neutron Reflectometry .................................................................................. 11
4.1 November 2013 ...................................................................................... 11
4.1.1 Experimental .................................................................................... 11
4.1.2 Results ............................................................................................. 12
4.2 Logbooks ................................................................................................ 15
4.2.1 November 2013 ................................................................................ 15
1 Summary and Discussion
First surface plasmon resonance (SPR), electrochemical impedance
spectroscopy (EIS), and neutron reflectometry (NR) experiments draw an
incoherent picture of the PlyC-B interaction with the lipid membrane. All
experiments were carried out using a 90:10 DOPC:DOPS tethered lipid
membrane (tBLM). SPR and EIS experiments were carried out, using a low-salt
buffer of an ionic strength of 40 mM Na+. NR experiments additionally used a
buffer with a physiological concentration of Na+ of 160 mM. SPR measurements
yielded a result, which suggest that upon PlyC-B incubation with concentrations
as low as 1nM, the bilayer degrades within seconds. The changes in the SPR
signal suggest the loss of several vol% of bilayer material. After that initial loss, a
partial recovery of the SPR signal takes place over minutes, which exhibits the
functional form of a binding process. EIS does not confirm the massive loss of
bilayer material observed with SPR. The defect density increases measurably
over the course of the SPR experiment, but the volume density of the observed
defects can be estimated to be below 1%. NR experiments confirm this result, as
bilayer properties remained unchanged within the resolution of the measurement
(1 vol%) over the entire measurement series. Only at the highest measured
concentration of PlyC-B (100 nM) and under low-salt conditions, a significant
1
binding of non-bilayer material was observed. Although the length of the
distribution of this material is comparable to the height of PlyC-B, the maximum
volume occupancy is very small with 3% only.
2 The PlyC-B protein
Figure 1: X-ray structure of PlyC-B, top and side view. The height of the protein along the z-axis is
about 20 Å.
3 SPR Experiments
Table1: List of SPR experiments.
Date
Experimental
Conditions
stBLM
Protein
05/12/2013
20 mM NaCl
20 mM NaH2PO4
at pH 7.4
90:10
DOPC:DOPS
PlyC-B
0.001µM-0.01µM
PBS buffer at pH
7.4 (8% glycerol)
90:10
DOPC:DOPS
PlyC-B
0.001µM-5µM
(not dialyzed)
Vesicle fusion
PBS at pH 7.4
90:10
DOPC:DOPS
PlyC-B
0.016µM-80µM
(dialyzed)
Rapid solvent
exchange
(RSE)
01/26/2014
01/31/2014
2
stBLM
preparation
Vesicle fusion
02/01/2014
PBS at pH 7.4
90:10
DOPC:DOPS
PlyC-B
0.016µM-80µM
(dialyzed)
Vesicle fusion
3.1 May 2013
Initial SPR experiment using a 90:10 DOPC:DOPS tBLM showed a change of the
SPR resonance angle upon incubation of with concentrations of PlyC-B as low as
1nM (see Figure 2 and Figure 3). The typical time-course of the measured
responses is an initial decrease of the SPR resonance angle, which is related to
a higher index of refraction of the material at the interface, commonly interpreted
as a loss of bilayer material and replacement with water. After that initial drop,
which occurs within seconds after injection, typically an incomplete recovery of
the resonance angle is observed, which can be interpreted by an addition of
material at the interface, bilayer or protein.
Figure 2 SPR curve for a sequential incubation of a 90:10 DOPC:DOPS tBLM with 10 nM and 100 nM
PlyC-B.
3
Figure 3: SPR curve for a sequential incubation of a 90:10 DOPC:DOPS tBLM with PlyC-B.
Impedance data (see Figure 4), taken before and after the SPR measurements
displayed in Figure 3, show an initial bilayer, which is low in defects. After the
SPR measurement, the bilayer shows a significantly lower resistance, but a lipid
bilayer is clearly still present.
Figure 4: Electrochemical Impedance of the tBLM before and after the SPR measurement shown in
Figure 3. The Cole-Cole plot indicates a well-insulated bilayer before the SPR measurements and a
defect-rich bilayer afterwards.
4
3.2 January 2014
3.2.1 Non dialyzed PlyC-B
The SPR experiment was conducted on a tBLM composed of DOPC:DOPS
90:10 (see Figure 5) prepared by vesicle fusion, which guarantees a better
control of the final composition of the lipid bilayer. The 64µl stock of PlyC-B was
thawed and diluted out to yield 12 concentrations ranging from 0.001µM to 5µM.
No dialysis was performed.
There is no observable change in the SPR signal when adding up to 300nM of
PlyC-B. This result is in agreement with the neutron measurements performed on
November 2013, where no protein was found at the interface up to a
concentration of 100nM. The dissociation equilibrium constant (Kd) couldn’t be
determined due the low concentration of the PlyC-B stock (15µM).
Figure 5: SPR raw data. DOPC:DOPS 90:10 tBLM was exposed to PlyC-B concentration series and
followed by a buffer rinse.
5
EIS measurements were carried out to assess the bilayer integrity before and
after the SPR (see Figure 6). In contrast with the May 2013 data sets, nearly any
change in the tBLM spectrum is noticeable.
Figure 6: EIS spectra before (black) and after (red) SPR experiment. The lipid bilayer electrically
sealing properties were conserved all the through the SPR experiment and no defect-rich region was
created.
After this experiment was performed, I learned later that the protein was stored in
PBS buffer containing 8% glycerol. In addition, the SPR curves do not show
saturation as the concentrations go higher which implies that glycerol might have
an effect on its own on the SPR signal. tBLM was then exposed to concentration
series of varying amounts of glycerol (0.5 to 3%) in order to test whether the
increase in pixels observed in Figure 5 was due to glycerol effect on the bulk or
due to protein adsorption (see Figure 7 and 8). Linear increase of pixel as a
function of glycerol concentration can be observed (inset of Figure 7). That is a
strong indication that glycerol is altering the refractive index of the bulk medium.
6
Figure 7: Glycerol effect on tBLMs. The SPR signal increases linearly with increasing percentages of
glycerol(inset), suggesting that glycerol affects the bulk refractive index.
The same experiment with glycerol was repeated but now a rinse step
immediately following the final concentration. The signal dropped back to the
baseline which confirms that the changes seen for the non-dialyzed protein can
be attributed, at least partially, to changes in the bulk refractive index rather than
to PlyC-B association.
Figure 8: Same experiment as Figure 7. After the rinse, the signal dropped back to the baseline.
Glycerol is not adsorbing to the tBLM, the increase in pixels is characterized solely by changes in
the bulk refractive index.
7
3.2.2 Dialyzed PlyC-B
Two SPR experiments were performed by exposing a tBLM composed of
DOPC:DOPS 90:10 to PlyC-B. The protein was dialyzed in order to get rid of the
8% glycerol and afterwards diluted to prepare 15 concentrations ranging from
0.016µM to 80µM(Figure 9). The first bilayer was completed by rapid solvent
exchange, which consists basically of flushing the organic lipid solvent with an
aqueous buffer. Consistently with the previous experiment, the bilayer integrity
was preserved throughout the measurements. No binding was detected up to
0.48µM. The result agrees with the neutron measurements taken on November
2013. The final PlyC-B concentration was followed by a rinse after which a small
fraction of PlyC-B stayed at the interface.
Figure 9: SPR raw data. Concentration series of dialyzed PlyC-B titrated on a DOPC:DOPS 90:10
tBLM.
The second experiment (see Figure 10) was carried out at the same
experimental conditions but vesicle fusion was used as the bilayer preparation
method instead of rapid solvent exchange. Once more there is no noticeable
binding up to 0.48 µM. Similarly to the first experiment, a small fraction of PlyC-B
remains on the membrane after rinsing. The raw SPR data shows a lower total
change in pixels even though the same concentration series was titrated.
8
Figure 10: SPR experiment was conducted at the same experimental conditions of experiment
shown in figure 9. Lipid bilayer was completed by vesicle fusion, though.
Just for consistency the bilayer was measured before and after the experiment.
Again, no change on the bilayer spectrum was observed (see Figure 10).
Figure 11: EIS data taken before (red) and after (black) the SPR experiment. The curves are
essentially overlapping.
9
High concentrations of PlyC-B might have an impact on the bulk refractive index.
I tried to estimate the contributions of bulk refractive index changes based on the
protein concentration, protein mass, and Sidd's work (Shenoy et al supporting
information from Membrane association of PTEN tumor suppressor).
Mass-weighted refractive indices of PBS buffer and PlyC-B mixtures at different
concentrations were calculated. The estimate predicts that a final concentration
of 13uM of PlyC-B contributes to 1 pixel change from the bulk medium to the
SPR signal. At 80uM the bulk contributions are as high as 6 pixels. For the
calculations, I assumed the refractive index (RI) of PlyC-B to be 1.41 and PBS
buffer to have the same RI as water 1.32972 (λ=763.8nm).
Figure 12: Binding curve obtained from the first SPR experiment of PlyC-B interacting with
DOPC:DOPS 90:10 tBLM. The red dots are the experimental data points. The blue triangles represent
the experimental data corrected for the bulk refractive index contributions.
10
Figure 13: Binding curve obtained from the second SPR experiment of PlyC-B interacting with
DOPC:DOPS 90:10 tBLM. The red dots are the experimental data points. The blue triangles represent
the experimental data corrected for the bulk refractive index contributions
The corrected data sets were fitted up to 24.1uM. This concentration was chosen
based on the raw SPR data. It reveals that subsequent PlyC-B concentrations
show no signs of saturation. Fits of both experiments predict Kd around 8uM.
The Bmax parameter, which corresponds to surface coverage, diverges for the 2
experiments. One possible explanation is the difference between the methods
used to prepare the bilayers, vesicle fusion in my opinion would be more reliable.
4 Neutron Reflectometry
4.1 November 2013
4.1.1 Experimental
The tethered lipid bilayer was prepared using vesicle fusion. The vesicle
suspension was concentrated at 10mg/mL and sonicated for 180 min in a 500
mM NaCl buffer. The HC18 SAM was incubated for 70 min and the incubation
solution was slowly replaced with 2x10 mL of 50 mM NaCl buffer. For neutron
11
measurements the buffer was replaced (see Table 2). Before use the 1mg/mL
stock solution of the 64kDa PlyC-B protein was divided into aliquots of 64 µL.
Table 2: Measurement conditions for NR.
File name
pl002-03
pl004-05
pl006-07
pl008-09
pl010-11
Condition
Neat bilayer, 70:30 β-mercaptoethanol :
HC18, 90 : 10 DOPC:DOPS
Buffer: 10 nM NaH2PO4, 150 nM NaCl,
pH 7.4
Incubating 1nM PlyC-B, diluted from
1mg/mL stock into the above listed buffer
Incubating 10nM PlyC-B, diluted from
1mg/mL stock into the above listed buffer
Incubating 100nM PlyC-B, diluted from
1mg/mL stock into the above listed buffer
Incubating 100nM PlyC-B, diluted from
1mg/mL stock into low-salt buffer
Buffer: 20 nM NaH2PO4, 20 nM NaCl, pH
7.4
Contrast
D2O, H2O
D2O, H2O
D2O, H2O
D2O, H2O
D2O, H2O
4.1.2 Results
The bilayer preparation yielded a complete bilayer and the thickness of the outer
lipid leaflet, and therefore the area per molecule, are consistent with literature
values. Protein addition did not significantly change bilayer parameters. Only the
measurement using 100 nM PlyC-B and low-salt buffer yielded a significant
amount of additional material that could be interpreted as protein. This material is
located in the center of the bilayer, it has maximum volume occupancy of about
3% only, and the distribution is about 15-20Å wide. This length is in agreement
with the height of the PlyC-B molecule.
Table 3: A selection of median fit values of relevant model parameters and their 68% confidence
intervals.
1 nM
salt
Lipid Bilayer
Tether
thickness
12
12.6±0.3
phys. 10 nM phys. 100 nM phys. salt
Salt
12.7±0.2
12.6±0.2
100
nM
low salt
12.7±0.2
Thickness
inner
lipid
leaflet
Thickness
outer
lipid
leaflet
Bilayer
Completeness
Protein
Volume
surface
density
of
protein
18.2±0.5
18.1±0.4
18.4±0.5
18.5±0.6
14.1±0.4
14.3±0.4
14.1±0.5
13.8±0.4
1.00±0.01
1.00±0.01
1.00±0.01
1.00±0.01
0.32±0.26
0.42±0.31
0.33±0.24
0.56±0.25
Figure 5: Molecular model of the tBLM while incubating with 10nM PlyC-B under low salt conditions.
13
Figure 6: Molecular model of the tBLM while incubating with 10nM PlyC-B under low salt conditions.
Figure 7: Molecular model of the tBLM while incubating with 100nM PlyC-B under low salt
conditions.
14
Figure 8: Molecular model of the tBLM while incubating with 100nM PlyC-B under low salt
conditions.
4.2 Logbooks
4.2.1 November 2013
15