HYDROPHOBIC CORE REGION (Membrane Structure)

A. Fatty Acyl Chain Order in Saturated Lipid Membranes

The hydrocarbon chains of the lipid bilayer are in a liquid-like state as evidenced by X-ray diffraction, electron spin resonance spectroscopy, and differential scanning calorimetry studies. A quantitative characterization of the hydrocarbon chain order in lipid bilayers by means of 2H-NMR became possible by selectively deuterating both fatty acyl chains in a lipid molecule. Measurement of the deuterium quadrupole splittings, Avq, allowed calculation of the order parameter of the C—D bond vector at each labeled carbon atom. The variation of the order parameter |SCD| with the position of the labeled carbon atom in the membrane is the so-called "order profile." An example is shown in Fig. 5 for a membrane composed of DPPC at temperatures T > 41° C (liquid crystalline phase). The segmental order parameters are approximately constant for the first nine chain segments, but decrease towards the central part of the bilayer. The chain ordering can be explained on the basis of the rotational iso-meric model for hydrocarbon chains. In the region of constant order parameters, trans-gauche isomerizations occur only in complementary pairs, leaving the hydrocarbon chains essentially parallel to each other. This leads to well-ordered bilayers with disordered hydrocarbon chains. The decrease of the order parameter in the central region is due to increasing contributions of gauche states. The total number of gauche iso-meric states was estimated to be between 3 and 6 per chain. The quantitative evaluation of the deuterium data further yields the thickness of the hydrocarbon region of the bilayer. For DPPC bilayers in the liquid crystalline phase, an average thickness of about 30 A and a thermal expansion coefficient of —2.510-3/K was derived, in good agreement with X-ray diffraction experiments. It should be noted that this approach is also valid for determining the bilayer thickness of highly unsaturated membranes. The DPPC order parameter profile (Fig. 5) serves as a "gold standard" for molecular dynamics simulations of bilayers. Similar order profiles have now been established for a large variety of pure lipid membranes as well as intact biological membranes.

FIGURE 2 Deuterium magnetic resonance spectra of sn-2 and sn-3 phosphatidylcholine bilayers deuterated at different positions (50 wt% lipid, 50 wt% H2O). (A) 1,2-dipalmitoyl-sn-glycero-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-glycero-2-phosphocholine [Seelig et al. (1980). Biochemistry 19, 2215); (C) 1,2-dipalmitoyl-sn-glycero 3-phosphocholine deuterated in both chains at the C-3' segment; (D) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine deuterated in both chains at the C-10' segment [Seelig and Seelig (1974).

B. Incorporation of c/s-Double Bonds

As mentioned before, almost all biological lipids contain unsaturated fatty acyl chains at the sn-2 position. The incorporation of the cis-double bond introduces a kink in the otherwise straight chain and reduces the gel-to-liquid crystal phase transition temperature by some 40°C compared to the saturated lipid. The influence of the cis-double bond was investigated in bilayers composed of 1-palmitoyl-2-oleoyl-3-sn-phosphocholine (POPC) by either labeling the saturated palmitoyl chain or synthesizing deuterated cis-unsaturated oleic acid. As seen in Fig. 5 and Fig. 6, the shape of the order profile of the palmitic acyl chain is similar to that observed for the fully saturated DPPC (Fig. 5), but the magnitude of the order parameters is distinctly smaller in the unsaturated system. This demonstrates that the presence of a cis-double bond causes a more disordered conformation of the hydrocarbon chains. Considering the relative flexibility within the palmitic acyl chain, the deuterium resonance data indicate a local stiffening of those segments which are located in the vicinity of the rigid cis-double bond. An increase in temperature leads to a further decrease of the order parameters.

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-glycero-3-phosphocholinedeuterated at the choline headgroup (a-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 a-segment; negative charges increase it [Seelig et al. (1987).

FIGURE 4 Variation of the quadrupole splittings of the a- and the p-segment in the headgroup of phosphatidylcholine bilayers upon binding of cationic (open symbols) and anionic (solid symbols) substances. The difference between the quadrupole splittings of the a-segmentwith, (Av(Xb)), and without (Av°) guest molecule is plotted versus that of the p-segment under identical conditions, (Av(Xb) — Av°)/Xb = m/, where Xb, is the mole fraction of bound guest molecule and i stands for a or p. Metal ions (□) [Altenbach and Seelig (1984). Biochemistry 23, 3913; Macdonald and Seelig (1987). Biochemistry26, 1231, 6292]; drugs (A) [Boulanger etal. (1981). Biochemistry20, 6824; Seelig etal. (1988). Biochim. Biophys. Acta 939, 267; Bauerle and Seelig (1991). Biochemistry 30, 7203]; amphiphilies (V) [Scherer and Seelig (1989). Biochemistry28, 7720]; peptides (O) [Beschiaschvili and Seelig (1991). Biochim. Biophys. Acta 1061, 78; Kuchinka and Seelig (1989). Biochemistry 28, 4216; Roux et al. (1989). Biochemistry 28, 2313; Spuhler et al. (1994). J. Biol. Chem. 269, 23904; Wieprecht etal. (2000). Biochemistry39, 442; Schote etal. (2000). Pharm. Res.]; electrically neutral detergent (*) [Wenkand Seelig (1997). Biophys. J. 73, 2565]; inorganic anion (■) [Macdonald and Seelig (1988). Biochemistry 27, 6769]; peptides (♦) [Schote etal. (2000). Pharm. Res.]; amphiphiles (▼) [Scherer and Seelig (1989). Biochemistry 28, 7720]; phospholipids (•) [Marassi and Macdonald (1992). Biochemistry 31, 10031; Scherer and Seelig (1989). Biochemistry28, 7720; Pinheiro etal. (1994). Biochemistry33, 4896]. The slope is characteristic for the sign of the electric charge and is m = —0.52 ± 0.01 for cations and m = —1.01 ± 0.05 for anions [cf. Scherer and Seelig (1989).

FIGURE 5 Order parameters |SCD | as a function of the labeled carbon atom for 1,2-dipalmitoyl-3-sn-phosphocholine (•) and for 1-palmitoyl-2-oleoyl-3-sn-phosphocholine (A) at 42°C. The sn-1 chains are labeled.

FIGURE 6 The effect of a cis- and a trans-double bond on the order parameter profile. Bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine labeled at different positions in the sn-1 (A) and sn-2 chain (□) measured at 27 C; 1-palmitoyl-2-elaidoyl-sn-glycero-3- phosphocholine labeled in the sn-2 chain (o) measured at40°C.

The 2H-NMR spectrum of POPC membranes deuter-ated at the C-9, and C-10 positions of the oleic acyl chain shows two quadrupolar splittings, the larger corresponding to the C-9 deuteron and the smaller to the C-10 deuteron (Fig. 6). The presence of two quadroupole splittings at the same rigid segments is caused by a tilting of the cis-double bond with respect to the bilayer normal which produces different orientations for the C-2H vectors of the 9- and 10-carbon atoms. The angle between the bilayer normal and the C=C bond vector was found to be 7to8°. The order parameter of 1-palmitoyl-2-elaidoyl-sn-glycero-3-choline deuterated at the C-9 and C-10 trans-double bond of the elaidic acid chain is also included in Fig. 6. Due to the symmetry of the trans-double bond the two C—D vectors at the C-9 and C-10 position make the same angle with the C=C vector axis. They give rise to the same quadrupole splitting, and the evaluation of the order parameter of the C=C axis is straightforward. Taking into account the different geometries, the molecular ordering and the angular fluctuations of the cis- and trans-double bonds are identical. In addition, there are no quantitative differences between sn-1 and sn-2 chain segments at this position in the bilayer. The segmental fluctuations around the bilayer normal thus only depend on the distance from the lipid-water interface but not on the specific segment geometry.

The effect of cis-unsaturation was also investigated for the glycosphingolipid N-(oleoyl-d33)galactosylceramide incorporated at low concentration into liquid crystalline liposomes composed of 1,2-dimyristocyl-3-sn-phosphatidylcholine (DMPC) and POPC using the perdeuterated oleoyl chain as the reporter element. The primary effect of cis-9,-10 unsaturation in glycosphin-golipids proved to be similar to that of cis-unsaturation in glycerolipids. It was further shown that the overall dynamics of N-(oleoyl)galactosylceramide in fluid phospholipid membranes was very similar to that of glycerolipids with comparable acyl chains.

Increasing sn-2 unsaturation from one to six double bonds in PC leads to an inhomogeneous disordering along the neighboring perdeuterated sn-1 chain. As a consequence, the effect of a temperature increase leading to a decrease in the average chain length is somewhat less pronounced in lipids with three or more double bonds in the sn-2 chain than in lipids with only one double bond.

C. Effect of Cholesterol on the Order and Motion of the Lipid Hydrocarbon Chains

The influence of cholesterol on the order and mobility of lipid bilayers was investigated with both selectively deuterated lipids and deuterated cholesterol. Addition of 50% cholesterol to DPPC and DMPC bilayers was shown to lead to an almost twofold increase of the quadrupole splitting of the labeled fatty acyl chain segment compared to that of a cholesterol-free bilayer. When [3-2H] cholesterol was added to a nondeuterated DPPC bilayer, again a large quadrupole splitting of the cholesterol probe was observed. Both probes lead to the conclusion that a high concentration of cholesterol induces an essentially all trans-conformation in those hydrocarbon chain segments which are in contact with the rigid steroid frame. This condensing effect of cholesterol was also observed in monolayer-and neutron-diffraction experiments.

The effect of cholesterol on the order parameter profile of individual fatty acyl chains in DPPC bilayers was simulated by means of molecular dynamics calculations as displayed in Fig. 7. A distinct plateau with an order parameter of the C—D bond vector of SCD = -0.4 was detected. This means that the hydrocarbon chains are almost fully extended and that the order parameter of the long molecular axis, Smol = -2 SCD, approaches its maximum value of Smol = 1.

In highly unsaturated lipid mixtures, typical for nerve and retinal membranes, cholesterol induces an increase in the order of both saturated and polyunsaturated hydrocarbon chains. However, the increase in order is about a factor of 2 smaller for polyunsaturated than for monounsaturated lipids.

FIGURE 7 Effect of cholesterol. Order parameter of the sn-2 chain in DPPC bilayers without (□) and with (■) 50 mol% cholesterol as function of carbon atom.

As far as lipid headgroups are compared, addition of cholesterol increases the chain order in the sequence 18:018:1 PS < 18:0-18:1 PC < 18:0-18:1 PE for the monoun-saturated lipid mixture and in the sequence 18:0-22:6 PS < 18:0-22:6 PE « 18:0-22:6 PC for polyunsaturated mixtures. The cholesterol-induced variation of order parameters as a function of the chemical nature of the lipid species suggests a cholesterol-induced formation of lipid microdomains with a headgroup- and fatty-acyl-chain-dependent lipid composition. In particular, under physiological conditions, the formation of PC-enriched mi-crodomains has been proposed in which the saturated sn-1 chain is preferentially oriented toward the cholesterol molecule. The lifetime of a lipid molecule in a given cluster, however, is less than 10-4 s, and the cluster radius is probably smaller than 25 nm.

In a natural membrane the effect of cholesterol is very similar as in model membranes. This was shown, for example, for Acholeplasma laidlawii membranes by the incorporation of perdeuterated and selectively deuterated fatty acids.