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The trisodium salt of dimyristoylphosphatidylinositol-4-phosphate (DMPI-4P) has been synthesised specifically deuterated at particular sites in the headgroup. These materials have been used in neutron diffraction experiments, which successfully located the position (depth) of each of these deuterated sites to within +/- 0.5 A in a mixed model membrane (a 1:1 molar mixture of DMPI-4P with dimyristoyl-phosphatidylcholine, DMPC, in the L alpha phase, hydrated to the level of 28 water molecules per lipid molecule). The diffracted intensities were measured at four different D2O/H2O ratios and six orders of diffraction were obtained. These data sets, in conjunction with computer modelling, have been used to determine the orientation of the inositol ring of DMPI-4P, localising each vertical H-H distance to within approximately +/- 0.03 A. The orientation of the inositol ring is found to be one in which the C5 hydroxyl is extended out into the aqueous medium. This is, therefore, the most accessible site for water-borne reagents. This may be significant for the important pathway leading from PI-4P to PI-4,5P2. On the assumption that the P/ODAG bond is orientated parallel to the bilayer normal, these results are consistent with two possible conformations for the portion of the headgroup connecting the diacylglycerol to the inositol ring. Distinction between these two is difficult, but one may be favoured since the other involves close atom-atom contacts.

Dimyristoylphosphatidylinositol (DMPI) has been synthesized with the appropriate natural stereochemistry and labelled with deuterium at specific sites in the D-myo-inositol headgroup. 2H-NMR spectroscopy of DMPI in its lamellar phase at a molar ratio of water-to-lipid RW/L of 129 and at 70 degrees C reveals quadrupolar splittings delta v of 3.83 and 2.17 kHz, respectively, for the five axially oriented C-D bonds and the single equatorially oriented C-D bond of the D-myo-inositol headgroup. Between RW/L ratios of 129 and 210 and between 30 degrees C and 80 degrees C the value of the ratio of these splittings delta nu ax/delta nu eq varies significantly (between 1.17 and 4.38). If it is assumed that, at a particular temperature, there is a single preferred orientation of the inositol headgroup, and that motion of the DPMI molecule establishes axial symmetry with respect to the bilayer normal then the ratio of these quadrupolar splittings can be used to impose constraints on that orientation. For example, the data are inconsistent with a situation in which the inositol ring lies parallel to the membrane surface and are difficult to reconcile with an arrangement where the inositol ring lies perpendicular to the surface. Computational modelling identifies four possible 'tilted' orientations, all of which are consistent with the data, and two of these allow good intramolecular hydrogen bonds to be formed. In one there is hydrogen bonding between the inositol C2-OH and the phosphate pro-R oxygen. This is close to the conformation previously identified as being dominant in DMSO solution (Bushby, R.J., Byard, S.J., Hansbro, P.M. and Reid, D.G. (1990) Biochim. Biophys. Acta 1044, 231-236).

The temperature dependence of the 1H-NMR spectrum of phosphatidylinositol (PI) in d6-dimethylsulphoxide (DMSO) shows that the hydroxy groups at C2 and at C6 of the inositol ring are internally hydrogen-bonded. This probably implies a trans/gauche conformation for the phosphate/inositol linkage. The presence of a trans phosphate-alkyl-oxygen bond is confirmed by 31P-NMR studies. If the conformation of PI in membranes is the same as that in DMSO solution, this implies that the inositol ring points out into the aqueous phase with its C1/C4 axis almost perpendicular to the membrane surface. Progress is also reported in attempts to characterise headgroup orientation and dynamics by 2H-NMR using deuterated synthetic PI, prepared by the route devised by Ward, J.G. and Young, R.C. (Tetrahedron Lett. 29 (1988) 6013-6016).