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HIGH PRESSURE X-RAY SURFACE DIFFRACTION ON HIGHLY ALIGNED LIPID SYSTEMS UNDER EXCESS WATER CONDITIONS.

H. Amenitsch(1), M.Kriechbaum(1), M. Steinhart(2), S. Bernstorff(3) and P. Laggner(1).


A hydrostatic high-pressure X-ray cell [1] was modified to perform studies on highly aligned lamellar lipid systems. With this experimental set-up at the SAXS-beamline at ELETTRA, Trieste, Italy, simultaneously wide- and small-angle X-ray scattering (SAXS) studies to elucidate the complex morphology of these systems were possible. In contrast to isotropic bilayer systems (usually liposomal preparations, also called "powder samples") highly aligned bilayer membrane systems give the possibility to derive additional structural information by diffraction. Aligned samples allow the clear differentiation of in-plane and out-of-plane correlation as well as the unique identification of the Miller indices in higher (>1) dimensional phases. However, aligning them under physiological conditions (i.e. excess water, ionic strength and physiological pH) has turned out to be a difficult task, e.g. the "vapor pressure paradox" [2, 3].
Using this modified high pressure cell, studies on these systems under physiological conditions, pressures between 0 - 3000 bar and temperatures between 0 and 80 C can be performed. Furthermore the cell offers the possibility to do p-jump experiments on these systems with jump amplitudes of up to 3 kbar/10 ms. In Fig.1 the 2-dimensional diffraction pattern of the aligned phospholipid (DMPC) obtained at 20 C and ambient pressure is shown. It clearly exhibits the in-plane reflection from the Pb phase after cooling down from the La phase, which is lamellar and has no in-plane reflections (see e.g. [4-6]). The real space model of the Pb phase is given in Fig.2. For the first time the set-up allowed to perform the La - Pb phase transition on aligned lipids using pressure as the thermodynamical variable increasing the pressure from 0 to 1080 bar at a temperature of 30 C. To compare the results the d-spacings obtained at different temperatures and pressures are compiled in Table 1.


Phase of DMPC Temperature (C) Pressure (bar) d-spacing (nm)
La 30 1 6.47
Pb cooling from 30C 20 1 7.06
Pb pressurizing from 1 bar 30 1080 7.38

Table 1. Liquid crystalline and stable ripple phase of DMPC.
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Figure 1. Diffraction pattern of a highly aligned lipid (DMPC) in the Pb phase at constant grazing angle at 20 C. Letter A shows the in-plane reflections due to the ripple structure (distance lambda), letter B gives the off-plane reflections due to the lamellar repeat distance d (see Fig.2). C denotes the specular reflectivity peak.
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Figure 2. Schematic sketch showing asymmetric rippled bilayer, separated by a layer of water. The unit cell (shown in red) is given by the dimensions d and lambda and the monoclinic angle gamma.
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References:
[1] Steinhart, M., Kriechbaum, M., Pressl, K., Amenitsch, H., Laggner, P. & Bernstorff, S. (1999), Rev.Sci.Instrum. 70, 1540-1545 .
[2] Rand, R.P., Parsegian, V.A. (1989), Biochim. Biophys. Acta 988, 351.
[3] Katsaras, J., Watson, M.J. (2000), Rev. Sci. Instrum. 71, 1737.
[4] Tardieu, A., Luzzati, V., Reman, F.C. (1973), J.Mol.Biol. 75, 711.
[5] Wack, D.C., Webb, W.W., (1989), Phys.Rev. A 40, 2712.
[6] Katsaras, J., et.al. (2000), Phys.Rev. E 61, 5668.

Author: Heinz Amenitsch
amenitsch@elettra.trieste.it (Sept.18th 2000)