31 P NMR in lipid membranes. CSA recoupling. Ludovic BERTHELOT , Dror E. WARSCHAWSKI & Philippe F. DEVAUX 1 1 Laboratoire de physico-chimie moléculaire des membranes biologiques UPR 9052 Introduction 31 P NMR experiments have been carried out with liposomes containing lipid mixtures or red blood cell membranes. We used MAS with a rotating speed of 5kHz, and recoupling of the CSA by rotation synchronized π-pulses. We have been able to separate the lipids on a 2D-spectrum according to their polar headgroup. The goal is to attribute the phase of each lipid by comparing the cross-sections of the spectrum with a static spectrum. Alpine conference on solid state NMR, Chamonix, 12-16 September 1999
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P in lipid membranes. CSA recoupling. · [3] Schmidt Rohr K. & Spiess H.W. “€Multidimensional solid-state NMR and polymers€”, 1994, Academic Press. [4] Tycko R., Dabbagh G.
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31P NMR in lipid membranes.CSA recoupling.
Ludovic BERTHELOT, Dror E. WARSCHAWSKI& Philippe F. DEVAUX 1
1 Laboratoire de physico-chimie moléculaire des membranes biologiques UPR 9052
IntroductionIntroduction
31P NMR experiments have been carried out withliposomes containing lipid mixtures or red blood cellmembranes. We used MAS with a rotating speed of5kHz, and recoupling of the CSA by rotationsynchronized π-pulses. We have been able toseparate the lipids on a 2D-spectrum according totheir polar headgroup. The goal is to attribute thephase of each lipid by comparing the cross-sections ofthe spectrum with a static spectrum.
Alpine conference on solid state NMR,Chamonix, 12-16 September 1999
Lipid Phases and NMRLipid Phases and NMR
• Structure of lipids investigated
• Lipid polymorphism in water : [1]
lamellarphase
invertedhexagonalphase (HII)
fluid Lα
gel Lβ
31P : natural abundance 100%I=1/2
O
O
O
O
O P
O
O
ON+_
O
O
O
O
O P
O
O
ONH3
_ +
O P
O
O
ONNH
OH
O
+_
PCPC
SMSM
PEPE
(phosphatidylcholin)
(phosphatidylethanolamine)
(sphingomyelin)
Static 31P NMR of phospholipids in waterStatic 31P NMR of phospholipids in water
ωω ωωββ
CS iso CSA== ++−−
∆ω∆ω3 1
2
2cos
for each phospholipid, theprecession frequency ωCS
depends on the orientation of thepolar headgroup with the field [2].
∆ω∆ωCSA chemical shift anisotropy
31P is in an anisotropic environment and undergoesrapid anisotropic motions.
The effective chemical shift tensor has acylindrical symmetry
$σσ
$σσσσ
σσσσ
==
⊥⊥
⊥⊥
0 0
0 0
0 0 //
ωω iso isotropic chemical shift
e.g. : static spectra ofDMPC at 10°C and 30°C
(gel-fluid transition : 23°C).
-40-30-20-1090 80 70 60 50 40 30 20 10 0 ppm
10°C
30°C
The result of the integration withrespect to β is characteristic of the lipid
phase [2] :
gel
fluid
inverted hexagonal
Micelles ororganic solvent
0 -100100
frequency (ppm)
Magic Angle SpinningMagic Angle Spinning
B0
ωωr/2ππ = 5kHz
ααm ααm = 54°44’
CSA is averaged outto zero
ωω ωω ωω ωωωω ωω
CS iso r r
r r
t C t C t
S t S t
( ) cos( ) cos( )
sin( ) sin( )
== ++ ++++ ++
1 2
1 2
2
2
for a rotation period of 200µs
ωω ωωCS iso==
Thus one obtains a high resolution spectrum, withone line for each headgroup.
The averaging is macroscopic instead ofmicroscopic.
2D recoupling2D recoupling
30°C
60°C
46°C
37°C
PCPE
recoupled indirect
dimension
decoupleddirect
dimension(MAS)
Correlation of a decoupleddimension (high
resolution, MAS) with arecoupled dimension [3] .
ωω ωω ωω1 1 2∝∝ == ++ ++CSstat
iso C C
We need :
so that the indirectdimension corresponds to a
static spectrum.
e.g. : static spectra of aPC/PE mixture at different
temperatures
A method derived from Tycko et al., 1989 [4]A method derived from Tycko et al., 1989 [4]
• Rotation synchronized ππ-pulses
(4 π-pulses)
We want :1
0 1 2tp t t dt C C
r
t
CSCSstat
iso
r
( ) ( ) ( )ωω χχ ωω χχ ωω∫∫ == == ++ ++
•p(t) is even
• p t t dt p t t dtt
r
t
r
trr r
( ) cos( ) ( ) cos( )ωω ωωχχ
0 0
12
12 2
2∫∫ ∫∫== ==
In fact : ωω ξξ ωω χχ ωωCS iso CSstatt( ) == ++
ξ ξ isotropic scaling factor
χχ anisotropic scaling factor
• The pulse program
+ 1H decoupling during the acquisition
π/2 π/2 π πππ
for τ1=39µs and τ2=89µs at 5000Hz,ξ = 0ξ = 0
χ = 0.393χ = 0.393
• no decoupling during the evolution time.
• no need for cross-polarization (31P is naturally
abundant).
• the direct dimension is proportional to a static
spectrum : the elements of the principal axis
tensor may be extracted directly.
• we just need a simple MAS probe to run our
experiments (no switch angle or speed).
Result #1DOPC/DOPE/cholesterol [5]
Result #1DOPC/DOPE/cholesterol [5]
vertical cross-
sections :
ωωr/2ππ=5000Hz ; χχ=0.393
37°C
PE PC
30°C
60°C60°C
46°C37°C
46°C
37°C
30°C
horizontal cross-section
of 2D
Result #2Ghosts of red blood cells
Result #2Ghosts of red blood cells
Hz
-2.5-2.0-1.5-1.0-0.52.0 1.5 1.0 0.5 0.0 ppm
-2000
-1500
-1000
-500
2000
1500
1000
500
0
PCPESM+
-12-10-8-6-4-214 12 10 8 6 4 2 0 ppm
PE SM+(0,60 ppm)
PC(0,00 ppm)
ωωr/2ππ=5000Hz ;χχ=0.393 ; 30°C
PCPS?
PE SM+
-2-12 1 0 ppm
MAS Hz5000
horizontal cross-sectionof 2D
vertical cross-sections : cholesterol 25
total phospholipids 56 in which PC 23
PE 20PS 11PI 2
SM 18others 1
average composition of lipids in humanerythrocytes (%)
ConclusionsConclusions
• a good signal/noise ratio even for biologicalsamples (experiment time : 3h30 ; ghosts :overnight).
• a good resolution in the direct dimension : lipidsare separated according to their polar headgroup.
• a quantitative narrowing of the recoupledspectrum with the temperature, corresponding tothe lamellar-hexagonal transition.
• recoupled spectra cannot besuperimposed on canonical staticspectra need to decouple duringmixing time, or to compare with staticnon decoupled spectra
-40-20100 80 60 40 20 0 ppm
static DMPC
spectrum
1H decoupled
non decoupled
• recoupled spectra for our ghosts do not exhibit aproper lineshape technical deficiencies (MASstability, amplifier power) ; probably also finitepulse length[6] and “ring down” effects.
[1] Cevc G. “ Phospholipids handbook “, 1993, Marcel Dekker,inc.
[2] Seelig J. “ 31P nuclear magnetic resonance and the headgroup structure of phospholipids in membrane ”, Biochim.Biophys. Acta, 1978, 515 : 105-140.
[3] Schmidt Rohr K. & Spiess H.W. “ Multidimensional solid-state NMR and polymers ”, 1994, Academic Press.
[4] Tycko R., Dabbagh G. & Mirau P.A. “ Determination ofchemical-shift-anisotropy lineshapes in a two dimensionalmagic-angle-spinning NMR experiment ”, J. Magn. Reson.,1989, 85 : 265-274.
[5] Moran L. & Janes N. “Tracking phospholipid populations inpolymorphism by sideband analyses of 31P magic anglespinning NMR”, Biophys. J., 1998, 75 : 867-879.
[6] Ishii Y. & Terao T. “Manipulation of nuclear spin hamiltoniansby rf-field modulations and its applications to observation ofpowder patterns under magic angle spinning”, J. Chem. Phys.,1998, 109 : 1366-1374.
ReferencesReferences
AcknowledgmentsAcknowledgments
This work was supported by CNRS. The authors wish to thank Pr.Geoffrey Bodenhausen and his whole team for fruitful discussions.