Multi-probe EPR study of a triglyceride containing phospholipid
membrane
Lars Duelund, Kirsi I. Pakkanen, John H. Ipsen
E-mail: [email protected]
MEMPHYS – Center for Biomembrane Physics, Department of Physics and Chemistry, University of Southern Denmark, Odense, Denmark.
Introduction
Effect of TO concentration on spectroscopic response
Cholestane label (CSL)
Triglycerides (TG) are the main components of many oils and is
generally the molecule used for long term energy storage in living
organisms. In mammals TG is usually stored in lipid droplets and
is transported between different cells and organelles in lipoprotein particles. Small amounts of TGs are also found i the cell
membrane of several different cells. Especially high amounts of
TGs are found in cancerous cells were they are suggested to be
found in small (Suggested to bee between 25 and 60 nm diameter)
droplets between two leaflets of the cell-membrane [1, 2].
Despite that TGs are found in cell membrane, very few studies
of the impact on TG on cell membranes or model membranes
exist. We have recently shown by range of different biophysical
techniques that the presence of around 5 mol% of triglyceride in
a phospholipid model membranes strongly modifies the spectroscopically reported properties of the membrane. We investigated
the effect of triolein (TO) in a model membrane of 1-Palmitoyl2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) (se fig 1) [3]
Figure 7: The
order parameter
of the CSL in the
POPC: TO 9:1
system. When we
compare the CSL
result with the
X-DSA and MeX-DSA results We
find an intermediate response,
which does not
allow clear-cut
conclusion about
the positioning of
the CSL probe.
Figure 4: The molecular order parameter,S, of 5PC as a function of TO added. Numbers are the mean of six samples ± 1
SE. Small letters represent statical significance.
The following points emerge:
The order parameter of the 5 PC probe is almost independent
of the amount of added TO.
Membrane Permeation of HBT
We observe a less ordered LF also for the pure POPC sample.
For the Me-5-DSA we see a strong and complex dependence
of S on the amount of added TO.
Figure 8: The
EPR spectra of
HBT in different
phases of the
POPC: TO 9:1
system. Interestingly we observe
a signal from an
HBT probe which
is in the aqueous
phase in the LF
phase.
Me-X-DSA vs. X-DSA
Figure 1: Structures of the used lipids and the structure of the
resulting multilamellar vesicle.
Figure 5: The molecular
order parameter, S, for
5-DSA and Me-5-DSA in
the different phases of the
POPC:TO 9:1 system.
I the earlier work [3] we observed that the POPC:TO 9:1 sample separated efficiently into two phases, both spontaneously in
normal gravity and in centrifugal field. We have labeled the
phases according to their sedimentation behaviour so that the upper phase was named light phase (LF) and the lower was named
heavy phase (HF). We also found that not all the added TO was
incorporated to the membranes, resulting in a TO concentration
of 5 mol% in both phases.
Conclusion & outlook
Spin labels with strong head-groups (5PC and X-DSA) show
a weak dependence on the presence of TO.
Figure 2: The system has been
investigated by molecular dynamics simulations [4]. In
this simulation snapshot of
the POPC:TO the clustering
of TO molecules in a intra
membrane blister is clearly
seen. In red TO, blue fatty
acid chains, dark green head
group, light green water.
Spin labels with weak head-groups (Me-X-DSA) show a
strong dependence on the presence of TO.
Can be result of different positioning of the probes. ie. the
Me-X-DSA is placed fully in the internal blister domain.
Figure 6: The correlation
time τc for 16-DSA and
Me-16-DSA in different
phases of the POPC:TO
9:1 system.
The τc was calculated as follows:
We have observed that the properties reported by EPR show a
strong dependence on the used probe. Therefore we have investigated as series of different spin labels, see fig 3 in a TO POPC
membrane.
−1/2
τc = kW0(h0/h−1 ) − 1) (1)
Were h0 is the line high of the
central line, h1 is hight if the
high field line and W0 the with
of the central line.
No clear conclusion on the positioning of the CSL is possible.
The presence of a signal from aqueous HBT in the POPC:TO
9:1 LF, and not in the HF, indicates a lower permeability in
the LF.
All findings need verification by further experiments. Especially determination of the position on the nitroxide ring relative to the membrane surface position by Gd+3 quenching
would be very relevant.
Acknowledgment
Dr. H. Khandelia is thanked for fig.2. MEMPHYS – Center for Biomembrane Physics is supported by the Danish National Research
Foundation.
When comparing Me-X-DSA with X-DSA the following point
emerges:
As with 5PC, there is almost no change in the observed order
parameter of X-DSA upon addition of TO.
The methylated probes show a strong change upon addition
of TO.
Figure 3: The used spin labels.
Interestingly the Me-5-DSA is mostly affected in the LF
where as the Me-16-DSA is mostly affected in the HF.
References
[1] C. Mountford, L. Wright, Organization of lipids in the plasma membranes of malignant and stimulated cells: a new model, Trends
in Biochemical Sciences 13 (5) (1988) 172–177.
[2] A. Ferretti, A. Knijn, E. Iorio, S. Pulciani, M. Giambenedetti, A. Molinari, S. Meschini, A. Stringaro, A. Calcabrini, I. Freitas,
et al., Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts, Biochimica
et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids 1438 (3) (1999) 329–348.
[3] K. Pakkanen, L. Duelund, M. Vuento, J. Ipsen, Phase coexistence in a triolein-phosphatidylcholine system. Implications for lysosomal membrane properties, Chemistry and physics of lipids 163 (2) (2010) 218–227.
[4] H. Khandelia, L. Duelund, K. Pakkanen, J. Ipsen, Triglyceride blisters in lipid bilayers: Implications for lipid droplet biogenesis
and the mobile lipid signal in cancer cell membranes, PLoS ONE Accepted.
Multi lamellar vesicles were prepared by mixing stock solutions of the appropriate lipids and spin labels in chloroform. The solvent was removed by a gentle stream of nitrogen and the sample was placed under vacuum overnight. The samples were hydrated with water to give final lipid concentration of 10 mM and 0.3 to 1 mol-% of the spin-label. Samples containing X-DSA spin labels were hydrated with a 1mM bicien buffer at
pH=9.06. Electron paramagnetic resonance spectroscopy was performed with a Bruker EMX Plus spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a PremiumX microwave bridge and Bruker internal variable temperature unit. For the measurement approximately 75 µl of the vesicles were transferred to a micro hematocrit tube which was left over night at 5◦ C, during which the heavy phase descended to the lower
part of the tube. The hematocrit tube was placed in a standard EPR tube, which was inserted into the spectrometer. For the measurement of the heavy phase the tube was adjusted so that we measured on the lower 1 cm of the tube. For the light phase the tube was adjusted so that the upper 1 cm of the tube was measured. All spectra obtained were X-band spectra (frequency ≈ 9.43 GHz) recorded with a power of 20 mW, a modulation
frequency of 100 KHz, modulation amplitude 1 G and time constant of 20.48 ms. Statistics The non-parametric Mann-Whitney U test was used to test statistical significance in the data. Confidence level was set to 0.05. Statistical analyses were made using R (R Development Core Team, 2005).
5th EF-EPR Summer School on Advanced EPR Spectroscopy, 5th – 12th September 2010 Konstanz, Germany.