The Membrane–Water Interface

The membrane–water interface comprises the lipid headgroup proper, with tightly bound hydration water and a more loosely packed extended hydration layer; the glycerol backbone; and the ester linkages of the fatty acyl chains. Due to conformational constraints, the carbonyl group of the sn-2 chain is particularly close to the lipid– water interface, while that of the sn-1 chain is inserted deeper into the membrane interior.

A. Headgroup and Glycerol Backbone Conformation of Phospholipids
The crystal structures of three synthetic lipids are shown in Fig. 1. Essential elements of these crystal structures are carried over into the liquid crystalline state as revealed by solid-state NMR. The main features are as follows: For PC, PE, and PG, the glycerol backbone is oriented perpendicular to the bilayer surface, while the polar headgroups are almost parallel to the membrane surface. Neutron scattering experiments of selectively deuterated lipid headgroups in liquid crystalline and gel state membranes determine the mean label position with an accuracy of up to ±1 Å and provide independent support for the almost parallel headgroup orientation of PC, PE, and PG.

The headgroup orientations of PC, PE, and PG bilayers in the liquid crystalline phase, in the gel phase, and in single crystals are thus very similar and independent of the dynamic state of the membrane. The correlation times of the segmental and collective motions of the head groups decrease abruptly by more than two orders of magnitude at the gel-to-liquid phase transition; nevertheless, the average conformation remains unaltered.

Phosphatidylserine measured at neutral pH and in the absence of ions is similar to the other phospholipids with respect to the glycerol backbone, but differs distinctly in its headgroup orientation and motion. The PS headgroup is rigid and exhibits little internal flexibility. A crystal structure is not available so far.

For the comparison ofNMRand X-ray diffraction measurements, the effect of membrane hydration can be relevant. A minimum of 11 to 16 water molecules per lipid molecule is needed to form a primary hydration shell for PC, PE, and PG. Additional water is in exchange with the primary hydration shell. With increasing hydration (10– 70 wt% H2O) the −P-N+ dipole of the phosphocholine headgroup was shown to move with its cationic end away from the hydrocarbon layer. This explains why the −P-N+ dipoles in liquid crystalline membranes are generally slightly tilted away from the membrane surface up to an angle of about 30°, while they are oriented parallel to the surface in the crystal structure.
Single-crystal structures of three phospholipids. The lipids are 1,2-dilauroyl-<em>sn-</emglycero-3- phosphoethanolamine (DLPE) [Hitchcock et al. (1974). Proc. Natl. Acad. Sci. USA 71, 3036], 1,2-dimyristoyl-snglycero- 3-phosphocholine (DMPC) [Pearson and Pascher (1979). Nature 281, 49], and 1,2-dimyristoyl-<em>sn-</emglycero- 3-phosphogycerol (DMPG) [Pascher et al. (1987). Biochim. Biophys. Acta 896, 77]. Structural features which are carried over into liquid-crystalline membranes: (1) the polar groups are oriented at approximately a right angle to the hydrocarbon chains, and (2) in DLPE and DMPC the <em>sn-</em2 fatty acid chain is bent at the C-2 segment while the <em>sn-</em1 chain is straight. A bent <em>sn-</em2 chain is a common property of phospholipids in biomembranes. Only one of two possible conformations is shown for each lipid [Seelig et al. (1987). Biochemistry 26, 7535].
Figure 1 Single-crystal structures of three phospholipids. The lipids are 1,2-dilauroyl-sn- sn- sn- sn- sn-

B. Ester Linkage of the sn-2 Fatty Acyl Chain Is Part of the Lipid–Water Interface
If the two fatty acyl chains of lipids are deuterated at methylene groups immediately next to the ester linkage they give rise to quite different quadrupole splittings, indicating that the beginnings of the two chains have different conformations. The inequivalence of the two chains was first observed for 1,2-dipalmitoyl-snglycero- 3-phosphocholine (DPPC) in its liquid crystalline phase. It is also preserved in the presence of transmembrane proteins in reconstituted and in natural membranes.

The conformational difference between the two fatty acyl chains is particularly pronounced for the C-2 segments (i.e., the segment next to the ester linkage) which give rise to three separate resonance splittings (cf. Fig. 2A). It is still detectable at position C-3 (Fig. 2C) but averages out at label positions deeper in the hydrocarbon layer (Fig. 2D, the C-10 segment).

At a molecular level, the different splittings indicate that the sn- sn-2 chain starts out parallel to the bilayer surface and makes a 90° bend after the C-2 segment in order to keep the sn-1 and sn-2 chains parallel to each other in agreement with the crystal structure shown for PC and PE in Fig. 1. Furthermore, labeling of the glycerol backbone suggests the possibility of two long-lived conformations of the glycerol constituent.

The conformation of the two fatty acyl chains near the glycerol moiety was further investigated by synthesizing specifically deuterated 1,3-dipalmitoyl-sn- sn-

One of the most extensively investigated proteins active at the membrane surface is phospholipase A2. This enzyme is water soluble, attacks the membrane from the aqueous phase, and acts specifically on the sn- sn-
Deuterium magnetic resonance spectra of <em>sn-</em2 and <em>sn-</em3 phosphatidylcholine bilayers deuterated at different positions (50 wt% lipid, 50 wt% H2O). (A) 1,2-dipalmitoyl-snglycero- 3-phosphocholine deuterated in both chains at the C-2´ segment [Seelig and Seelig (1975). Biochim. Biophys. Acta 406, 1]; (B) 1,3-bis-([2 ,2 -2H2]palmitoyl)-<em>sn-</emglycero-2-phosphocholine [Seelig et al. (1980). Biochemistry 19, 2215); (C) 1,2-dipalmitoyl<em>sn-</em glycero 3-phosphocholine deuterated in both chains at the C-3´ segment; (D) 1,2-dipalmitoyl-<em>sn-</emglycero-3-phosphocholine deuterated in both chains at the C-10´ segment [Seelig and Seelig (1974). Biochemistry 13, 4839].
Figure 2 Deuterium magnetic resonance spectra of sn- sn- 2O). (A) 1,2-dipalmitoyl-snglycero- 3-phosphocholine deuterated in both chains at the C-2´ segment [Seelig and Seelig (1975). Biochim. Biophys. Acta 406, 1]; (B) 1,3-bis-([2 ,2 -2H2]palmitoyl)-sn- sn-sn-

C. Phospholipid Headgroup Response to Ions
The quadrupolar splitting, ΔνQ, of headgroup-deuterated PC or PE varies linearly as a function of the total amount of electric surface charge, and changes in opposite directions are induced by positive and negative surface charges. This indicates that the phosphocholine and the phosphoethanolamine dipoles are sensitive to electric charges at the membrane surface and function as an electrometer. As an example, the interaction of α-CD2-POPC with an anionic and a cationic amphiphile is shown in Fig. 3.

The chemical nature of the ion imparting the membrane surface charge appears to be of secondary importance. A variety of chemically different, charged compounds including metal ions, local anesthetics, peptides, hydrophobic cations and anions, amphiphiles, and lipids have been shown to yield similar results when incorporated as guest molecules into PC or PE membranes. This is demonstrated in Fig. 4, which summarizes the experimental results in a rather condensed representation. If a guest molecule is added to a headgroup deuterated phospholipid, the α- and β-quadrupole splittings (Δνα,Δνβ ) change linearly with increasing concentration. In Fig. 4 the slopes, mα and mβ , of such Δνα and Δνβ versus concentration plots are shown. A linear correlation exists between mα and mβ , with a slope of −0.5 for cations and −1 for anions. The molecular interpretation is as follows: A positive electric charge on the membrane surface surface moves the N+ end of the headgroup −P-N+ dipole away from the membrane surface, and a negative charge moves the N+ end towards the hydrocarbon phase. The out-of-plane movement of the phospholipid headgroup dipole creates a local electric field across the membrane, which can easily reach a field strength of 105 V/cm. Such high electric fields can, in principle, entail conformational changes of membrane-bound proteins, and the lipid dipole field could thus play a regulatory role in membrane function.

If the membrane contains negatively charged lipids to begin with, the concentration of cationic compounds at the membrane surface is drastically enhanced, facilitating the binding and also providing an additional mechanism of electric modulation.
Charged amphiphiles in lecithin membranes induce a reorientation of the −P-N+ dipole. The figure shows deuterium NMR spectra of 1-palmitoyl-2-oleoyl-<em>sn-</emglycero-3-phosphocholine deuterated at the choline headgroup (α- CD2-POPC) without amphiphile (Xb =0), with cationic amphiphile (Xb =0.14), and with anionic amphiphile (Xb =0.4). Positive charges decrease the quadrupole splitting of the α-segment; negative charges increase it [Seelig et al. (1987). Biochemistry 26, 7535].
Figure 3 Charged amphiphiles in lecithin membranes induce a reorientation of the −P-N+ dipole. The Figure shows deuterium NMR spectra of 1-palmitoyl-2-oleoyl-sn- 2-POPC) without amphiphile (Xb =0), with cationic amphiphile (Xb =0.14), and with anionic amphiphile (Xb =0.4). Positive charges decrease the quadrupole splitting of the α-segment; negative charges increase it [Seelig et al. (1987). Biochemistry 26, 7535].

D. Headgroup Orientation in Glycolipids and Glycosphingolipids and Their Influence on Phospholipid Headgroups
The deuterium order parameter of headgroup-labeled glycolipids and glycosphingolipids generally show a headgroup orientation in which the sugar residues project essentially straight up from the bilayer surface into the aqueous region, permitting maximum hydration of the glucose hydroxyl groups by water. The glucosyl headgroup appears to be rather rigid, but rotates with a rotational diffusion constant of ∼108 s−1.

The headgroup conformational changes of deuteriumlabeled PC observed in the presence of glycolipids and glycosphingolipids were shown to be qualitatively similar to those of negatively charged ions (cf. Fig. 4). However, in comparison to the effects induced by charged substances, these effects were modest.