P NMR in lipid membranes. CSA recoupling.

Transcription

1 31 P NMR in lipid membranes. CSA recoupling. Ludovic BERTHELT, Dror E. WARSCHAWSKI & Philippe F. DEVAUX 1 1 Laboratoire de physico-chimie moléculaire des membranes biologiques UPR 9052 Alpine conference on solid state NMR, Chamonix, September 1999 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.

2 Lipid Phases and NMR Structure of lipids investigated 31 P : natural abundance 100% P _ I=1/2 N + PC (phosphatidylcholin) PE (phosphatidylethanolamine) P _ + NH 3 H NH P _ + N SM (sphingomyelin) Lipid polymorphism in water : [1] lamellar phase fluid L α gel L β inverted hexagonal phase (H II )

3 Static P NMR of of phospholipids in in water 31 P is in an anisotropic environment and undergoes rapid anisotropic motions. The effective chemical shift tensor cylindrical symmetry $s has a $s = s ^ s 0 ^ Ł 0 0 s // ł w = w + Dw CS iso CSA 2 3cos b for each phospholipid, the precession frequency ω CS depends on the orientation of the polar headgroup with the field [2]. Dw CSA chemical shift anisotropy w iso isotropic chemical shift

4 The result of the integration with respect to β is characteristic of the lipid phase [2] : gel 10 C fluid 30 C inverted hexagonal ppm e.g. : static spectra of DMPC at 10 C and 30 C (gel-fluid transition : 23 C). Micelles or organic solvent frequency (ppm)

5 Magic Angle Spinning B 0 a m a m = w r /2p = 5kHz w ( t) = w + C cos( w t) + C cos( 2w t) CS iso 1 r 2 r + S sin( w t) + S sin( 2w t) 1 r 2 r CSA is averaged out to zero w CS = w iso for a rotation period of 200µs Thus one obtains a high resolution spectrum, with one line for each headgroup. The averaging is macroscopic instead of microscopic.

6 2D recoupling Correlation of a decoupled dimension (high resolution, MAS) with a recoupled dimension [3]. recoupled indirect dimension decoupled direct dimension (MAS) We need : stat w wcs = w iso + C + C so that the indirect dimension corresponds to a static spectrum. PE PC 30 C 37 C e.g. : static spectra of a PC/PE mixture at different temperatures 46 C 60 C

7 A method derived from Tycko et et al., 1989 [4] [4] Rotation synchronized p-pulses (4 π-pulses) We want : 1 t r t 0 r stat p( t) w ( t) dt = c w = c ( w + C + C ) CS CS iso 1 2 p(t) is even 1 t 2 r 2 r p( t) cos( w rt) dt = p( t) cos( 2w rt) dt = t c t 2 r In fact : w ( t) = x w + c w stat CS iso CS x isotropic scaling factor c anisotropic scaling factor

8 The pulse program for τ 1 =39µs and τ 2 =89µs at 5000Hz, x = 0 c = π/2 π/2 π π π π + 1 H decoupling during the acquisition no decoupling during the evolution time. no need for cross-polarization ( 31 P 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).

9 Result #1 DPC/DPE/cholesterol [5] [5] vertical crosssections : 37 C PE 30 C 37 C 46 C 60 C w r /2p=5000Hz ; c=0.393 PC horizontal cross-section of 2D 30 C 37 C 46 C 60 C

10 Result #2 Ghosts of of red blood cells Hz PE+ SM PS? PC MAS 5000Hz horizontal cross-section of 2D PE +SM PC ppm ppm w r /2p=5000Hz ; c=0.393 ; 30 C vertical crosssections : cholesterol 25 total phospholipids 56 in which PC 23 PE 20 PE+ SM (0,60 ppm) ppm PC (0,00 ppm) PS 11 PI 2 SM 18 others 1 average composition of lipids in human erythrocytes (%)

11 Conclusions a good signal/noise ratio even for biological samples (experiment time : 3h30 ; ghosts : overnight). a good resolution in the direct dimension : lipids are separated according to their polar headgroup. a quantitative narrowing of the recoupled spectrum with the temperature, corresponding to the lamellar-hexagonal transition. recoupled spectra cannot be superimposed on canonical static spectra need to decouple during mixing time, or to compare with static non decoupled spectra 1 H decoupled static DMPC spectrum non decoupled ppm recoupled spectra for our ghosts do not exhibit a proper lineshape technical deficiencies (MAS stability, amplifier power) ; probably also finite pulse length [6] and ring down effects.

12 References [1] Cevc G. Phospholipids handbook, 1993, Marcel Dekker, inc. [2] Seelig J. 31 P nuclear magnetic resonance and the head group structure of phospholipids in membrane, Biochim. Biophys. Acta, 1978, 515 : [3] Schmidt Rohr K. & Spiess H.W. Multidimensional solidstate NMR and polymers, 1994, Academic Press. [4] Tycko R., Dabbagh G. & Mirau P.A. Determination of chemical-shift-anisotropy lineshapes in a two dimensional magic-angle-spinning NMR experiment, J. Magn. Reson., 1989, 85 : [5] Moran L. & Janes N. Tracking phospholipid populations in polymorphism by sideband analyses of 31 P magic angle spinning NMR, Biophys. J., 1998, 75 : [6] Ishii Y. & Terao T. Manipulation of nuclear spin hamiltonians by rf-field modulations and its applications to observation of powder patterns under magic angle spinning, J. Chem. Phys., 1998, 109 : Acknowledgments This work was supported by CNRS. The authors wish to thank Pr. Geoffrey Bodenhausen and his whole team for fruitful discussions.