Fluidity and polarity of HIV Virion Mimic Membranes
defined by EPR spin-labeling methods
Jessica Saintibert1, Lu Yu2 and Likai Song2
1Tallahassee Community College, 444 Appleyard Drive Tallahassee, FL 32304
2 National High Magnetic Field Laboratory, Tallahassee, FL 32310
Lipid polarity
5-Doxyl in HIV
5-Doxyl
PC
mimic membrane
2Azz
16-Doxyl in HIV
16-Doxyl
PC
mimic membrane
Temperature
200K
3280
3300
3320
3340
3360
3380
Field (Gauss)
•  Magnetic tensor Azz was used to determine lipid polarity differences.
Larger 2Azz values corresponds to higher polarity.
•  The polarity of 5-Doxyl PC is higher than deeply buried 16-Doxyl PC.
•  Cholesterol increases the polarity of 5-Doxyl PC, but not 16-Doxyl
PC.
Methods
Lipid fluidity
•  EPR:
EPR spectroscopy was performed on a Bruker E680
spectrometer. EPR spectra were recorded at 2 mW incident
microwave power with a field modulation of 1.0 G at 100 kHz
using a Bruker High Sensitivity resonator.
• Membranes: HIV-1 virion mimic membranes were prepared
using DOPC/Sphingomyelin/DOPE/DOPG/Cholesterol with
the molar ratio of 9:18:19:9:45 [3].
2AA
5-Doxyl PC
16-Doxyl PC
2A//
45% Cholseterol
0% Cholseterol
2A//
16-Doxyl in HIV
mimic
membrane
16-Doxyl
PC
B
Temperature
293K
3320
3340
3360
3380
Field (Gauss)
•  16-Doxyl PC is more mobile than 5-Doxyl PC in cholesterol binding domain.
•  Cholesterol decreases the lipid fluidity of both 5-Doxyl PC and 16 Doxyl
PC.
•  EPR parameters A and B were used to determine lipid fluidity, e.g. order
parameter and the amplitude of motion (cone angle).
Lipid fluidity vs temperature
40
10
4
0
•  HIV virion membranes consist of phospholipid
bilayers and 45% of cholesterol. To be noted,
cholesterol positions in the lipids are tentative.
and 16-Doxyl PC) were used to determine membrane
fluidity (mobility) and polarity.
-4
-10
3280
3300
0.6
5-Doxyl, 0% Chol
5-Doxyl, 45% Chol
0.4
16-Doxyl, 0% Chol
16-Doxyl, 45% Chol
0.2
0
-­‐10
0
10
20
30
40
50
Temp. (OC)
Order parameter is varied between 0 (disordered) to 1 (ordered).
Amplitude of motion
90
60
5-Doxyl, 0% Chol
5-Doxyl, 45% Chol
16-Doxyl, 0% Chol
30
16-Doxyl, 45% Chol
-­‐10
20
•  Spin-labeled lipids at specific positions (5-Doxyl PC
0.8
0
2AA
3300
Cholesterol
B
5-Doxyl in HIV
5-Doxyl
PC
mimic
membrane
Lipid Bilayer
Lipid
Bilayer
45% Cholseterol
0% Cholseterol
Order parameter
2Azz
Since the acquired immunodeficiency syndrome (AIDS)
was recognized in 1981, an estimated 65 million infections
and 25 million deaths have been ascribed to human
immunodeficiency virus-1 (HIV-1) (CDC, 2006). Preventive
vaccination and effective treatment are paramount to
eliminate further global HIV-1 spread [1, 2]. The lipids of
HIV viruses play critical roles in viral infection, and are
the current target for vaccine and therapeutic design.
Here, EPR spin labeling techniques were used to define
the physical properties of HIV virion mimic membranes,
including lipid fluidity and polarity. Since the HIV
membranes consist of a mixture of different
phospholipids and cholesterol, the effect of cholesterol
on HIV membrane properties was also characterized.
Order parameter
1
Cone angle (degree)
Introduction
3320
3340
Field (Gauss)
3360
3380
EPR spectra
vs
temperature
0
10
20
30
40
50
Temp. (OC)
Conclusions
•  Adding cholesterol decreases lipid mobility.
•  Low polarity was found for deeply buried 16-Doxyl.
•  The HIV membrane cholesterol-binding-domain (CBD)
is near 5-Doxyl, and distant from 16-Doxyl.
•  Cholesterol increases the polarity of the CBD.
•  Lipid fluidity increases (order parameter and cone
angle) with higher temperature. The phase transition
temperature of HIV membrane CBD is around 0 to 4
OC.
Acknowledgements
I would like to express gratitude to Dr. Likai Song and his graduate
student Lu Yu for their help during this summer REU program.
Furthermore appreciation goes towards Jose Sanchez and his staff
for selecting me to be a part of this research experience. As well as
an immense thanks to Dr. Calandra Stringer for informing that a
program like this exists for undergraduates with STEM majors.
This work was supported by the NSF Award No. DMR 0654118.
References
1.  Song et al. (2009). Broadly neutralizing anti-HIV-1 antibodies
disrupt a hinge-related function of GP41at the membrane
interface. PNAS, 106 (22), 9057-9062
2.  Kim et al. (2011). Antibody mechanics on a membrane-bound HIV
segment essential for GP41-targeted viral neutralization. Nature
Struct. Mol. Biol., 18 (11), 1235-43
3.  Brügger et al. (2006). The HIV lipidome: A raft with an unusual
composition. PNAS, 103 (8), 2641-2646