P

di(C14:0)PC

Fig. 6.2. Headgroup conformations ofphosphatidyiethanoiamine [di(C12:0)PE] and phosphatidylcholine [di(C14:0)PC; molecule 2 in the unit cell]. The three glycerol carbons are marked sn-1 to sn-3. Coordinatesfrom Harlos et al. (1984) and Pearson and Pascher (1979)

diester group is such that the initial part of the sn-2 fatty acyl chain extends parallel to the bilayer surface but then bends sharply at the second carbon atom (Fig. 6.2). As a consequence, the sn-1 chain extends further into the bilayer than the sn-2 chain. The axial displacement of the two chains, 3.7 A, is equivalent to three methylene groups. In natural lipids, this difference is minimized since longer fatty acyl chains are normally found at the sn-2 position rather than at the sn-1 position. The glycerol backbone is oriented almost perpendicular to the bilayer surface with the sn-1 chain continuing in this direction forming an antiplanar zigzag chain together with the glycerol moiety (Fig. 6.2).

In the crystal of di(C12:0)PE the headgroups are arranged in antiparallel rows, with hydrogen bonds between the nitrogen atoms and the non-esterified phosphate oxygen atoms of neighbouring molecules (Pascher et al. 1992). The small separations between the nitrogen and oxygen atoms (2.7-2.9 A) have the character of both hydrogen bonds and ionic interactions. The structure of the di(C14:0)PC dihydrate is more complex (Pearson and Pascher 1979). The molecular area occupied by di(C14:0)PC is the same as that occupied by di(C12:0)PE (39 A2), but this is too small for the more bulky phosphorylcholine headgroup. The phosphatidylcholine molecules therefore pack in pairs, mutually displaced in the direction perpendicular to the bilayer surface. This means that the effective surface area per molecule is kept small. The headgroup conformations of the two crystallographically independent molecules making up each pair are very similar, being approximately mirror images of each other. Hydrogen bonding of the type observed in phosphatidylethanolamine between the ammonium and phosphate groups is not possible with the -N(CH3)3+ group in a phosphatidylcholine. Indeed, because of the bulky nature of the choline group, the nitrogen atom comes no closer than 4.5 A to the phosphate oxygens. Instead, the negatively charged phosphate groups are separated and shielded by water molecules of hydration. In the dihydrate, two water molecules are located between neighbouring phosphate ester groups, forming an infinite hydrogen bonded ribbon: phosphate(l)-water-phosphate(2)-water. Lateral interactions between phosphatidylcholine molecules can thus be expected to be weaker than those between molecules of phosphatidylethanolamine.

Very similar backbone and headgroup structures have been suggested from NMR and neutron diffraction studies for phosphatidylethanolamines and phosphatidylcholines in liquid crystalline phase bilayers in the presence of excess water. The conformational inequivalence of the two chains is maintained, with the glycerol backbone lying parallel to the bilayer normal (Seelig and Seelig 1975; Zaccai et al. 1979), although the chain equivalence is reduced compared to that in the crystal, and the terminal methyl ends of the two chains are out of step by only about 1.8 A (Zaccai et al. 1979). X-ray diffraction studies of di(C18:l)PC and a brominated analogue show that, in the liquid crystalline phase, the positions of the double bonds in the sn-l and sn-2 chains, when projected onto the bilayer normal, can be no more than 1 A apart (Wiener et al. 1991).

Studies using neutron scattering have shown that the lipid headgroups in phosphatidylcholines and phosphatidylethanolamines are oriented roughly parallel to the bilayer surface in the liquid crystalline phase in excess water (Buldt and Wohlgemuth 1981), as in the crystal structures. These headgroup structures represent average orientations, since molecular dynamics simulations show that there is considerable motion in the headgroup region of the bilayer, the orientation of the P-N vector in a phosphatidylcholine, for example, varying, for individual molecules, from an angle of zero with respect to the bilayer normal, so that the NMe3+ group is pointing out into the solvent, to values greater than 90°, so that the NMe3+ group is pointing into the hydrocarbon core of the bilayer (Heller et al. 1993; Stouch et al. 1994; Hyvonen et al. 1997). The conformation of the phospho-rylcholine headgroup is affected by the charge on the membrane, the headgroup acting as a molecular voltmeter (Seelig et al. 1987). Incorporation of positive charge into the membrane results in repulsion of the positively charged choline group, with a tilting of the "P-N+ dipole away from the surface of the membrane; conversely, introduction of negative charge attracts the choline group, pulling the "P-N+ dipole towards the surface (Seelig et al. 1987).

For phosphatidylserines, the glycerol backbone and headgroup structures are dependent on pH (Sanson et al. 1995). At acid pH, when the phosphatidyl-serine headgroup is zwitterionic with no net charge, the glycerol backbone conformation is very similar to that in a phosphatidylcholine or phosphatidylethanolamine, lying parallel to the bilayer normal. However, this changes at neutral pH, when the headgroup becomes negatively charged; the glycerol backbone is now oriented perpendicular to the bilayer normal, the conformation observed for negatively charged phospholipids in crystals. Whereas the headgroup lies parallel to the bilayer surface at acid pH (again as for phosphatidylcholine or phosphatidylethanolamine), at neutral pH it is more extended; this increases the distance between the serine carboxylate group and the layer of negatively charged phosphate groups, minimizing electrostatic interactions.

The ceramide backbone of glycosphingolipids in liquid crystalline bilayers also adopts a structure with a sharp bend in the fatty acyl chain away from the bilayer surface at C2, comparable to the bend in the sn-2 chain of the phosphatidylcholines (Johnston and Chapman 1988). However, it has been suggested that dialkylphospholipids adopt a structure in the liquid crystalline state in which the sn-2 chain is fully extended with no bend at C2 (Lohner 1996); the presence of two ester groups (in the diacylphospholipids and diacylglycerol) or an ester and an -OH group (in the glycosphingolipids) would seem to determine packing in the lipid backbone region of the bilayer. Despite the differences in the glycerol backbone region for the ester and ether linked phosphatidylcholines, the time-averaged conformations of the phosphorylcholine headgroups are the same (Paltauf 1994).

Our knowledge of the water structure close to a lipid bilayer surface is rather limited. This is unfortunate since interactions between lipid headgroups and water is likely to have a major effect on packing in the headgroup region, and possibly on interactions with membrane proteins. There are three regions in a phospholipid headgroup that are likely to be involved in interaction with water: the ester oxygens, the phosphate oxygens, and the carboxyl, amino, or tetramethylammonium groups of phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, respectively, although interaction of water with the tetramethylammonium group may be relatively weak. The extent of hydration is very different for phosphatidylcholines and phosphatidylethanolamines. At full hydration, a bilayer of dipalmitoylphosphatidylcholine [di(C16:0)PC] takes up about 23 molecules of water per molecule of lipid (Nagle and Wiener 1988), whereas a bilayer of di(C12:0)PE takes up only about 10 molecules of water per molecule of lipid (Mcintosh and Simon 1986). The first few water molecules to bind, bind tightly to the phosphate group and are motionally restricted (Volke et al. 1994). Further water molecules, although distinct from bulk water, are in fast exchange with bulk water. Measurements of NMR spin lattice relaxation times for deuterated water as a function of lipid hydration suggest that between about 11 and 16 water molecules occupy the first hydration shell around a phosphatidylcholine headgroup (Borle and Seelig 1983). In a molecular dynamics simulation of di(C16:0)PC, an average of 10.2 water molecules were found hydrating the -NMe3+ group in a clathrate-like cluster, 4.0 water molecules hydrating the phosphate, and one water molecule hydrating a carbonyl group (Marrink and Be-rendsen 1994). NMR studies, however, suggest, at least at low temperatures, that only five water molecules are needed to form a solvation shell around the -NMe3+ group (Hsieh and Wu 1997).

Molecular dynamic simulations of bilayers of di(C14:0)PC show that the head-groups are linked into clusters by water molecules and by charge interactions between the positively charged choline groups of one di(C14:0)PC molecule and the phosphate or carbonyl oxygen atoms of another molecule (Pasenkiewicz-Gierula et al. 1999). Strong intermolecular interactions between choline and phosphate groups on neighbouring molecules has been demonstrated in 31P-1H nuclear Overhauser experiments on phospholipid bilayers (Yeagle 1978) and molecular dynamics simulations suggest that intermodular charge interactions are much more important than intramolecular interactions between the choline and phosphate and carbonyl oxygens on the same molecule (Pasenkiewicz-Gierula et al. 1999). As well as salt bridges, the headgroups are linked by multiple water bridges, the water molecules hydrogen bonding mostly to the phosphate and carbonyl oxygens (Pasenkiewicz-Gierula et al. 1999). Individual water molecules exchange in and out of the lipid clusters relatively rapidly. The presence of an unsaturated fatty acyl chain increases the area occupied by the phospholipid molecule and increases the distance between the headgroups, leading to less interaction between the headgroups and increased accessibility of carbonyl and phosphate oxygens to water, with increased hydration of the lipid headgroup (Murzyn et al. 2001).

The pattern of hydration observed in simulations of a bilayer of a phosphati-dylethanolamine was distinctly different to that observed for a phosphatidylcholine (Damodaran and Merz 1994; Zhou and Schulten 1995). Whereas the hydrophobic -NMe3+ group induced formation of a clathrate-like hydration shell around the headgroups in order to optimize interwater hydrogen bonding, direct hydrogen bonds are formed between the -NH3+ group and the water molecules. Although interlipid hydrogen bonds are observed in the headgroup region of the phosphatidylethanolamine, more hydrogen bonds are formed with water; the hydrogen bonding interlipid network is much weaker than that observed in the crystal (Zhou and Schulten 1995).

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