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Angle from bilayer normal

Fig. 2. Increased acyl chain unsaturation and increased temperature produce dissimilar increases in acyl chain disorder. (A) Difference order-parameter profiles, AS(n), of deuterium NMR measurements on perdeuterated 18:0 in the sn-1 position as a function of changes in temperature and unsaturation at the sn-2 position. •: AS(n) between 18:0 , 22:6 PC and 18:0 , 18:1 PC; 0:AS(n) for 18:0^,18:1 PC between 27°C and 47°C. Carbon atoms are numbered beginning at the glycerol backbone. (Data from Gawrisch and Holte, 1996; used by permission of K. Gawrisch). (B) Difference in orientation probability for the fluorescent membrane probe DPH. Orientation distribution of DPH in 16:0,18:1 PC at 40°C minus that in 20°C (—), and the distribution of di22:6 PC minus that of 16:0,18:1 PC at 20°C (---).

in the terminal half of the acyl chain, whereas the disorder induced by raising the temperature occurs over the entire chain.

Elevated temperature and increased unsaturation also have distinct effects on the average acyl chain packing. DPH orientation difference curves that compare the DPH orientation probability in 16:0,18:1 PC between 20°C and 40°C, and between 16:0,18:1 PC and di22:6 PC are shown in Fig. 2B. The solid curve shows that raising the temperature reduces the population oriented parallel to the acyl chains and increases the population in the bilayer midplane, oriented parallel to the bilayer surface. This shift indicates that an increase in temperature alters acyl chain packing in such a way that the bilayer mid-plane can accommodate a larger fraction of the DPH molecules. However, the distributions remain fairly narrow. The region below the zero line in the dashed curve shows that the presence of 22:6n-3 acyl chains also reduces the DPH population oriented parallel to the acyl chains. However, the dashed curve above the zero line shows that the presence of 22:6n-3 acyl chains produces a pronounced broadening of both orientational modes, rather than just a shift of DPH from the parallel mode to the perpendicular mode. The two comparisons in Fig. 2 demonstrate that elevated temperature and increased acyl chain unsaturation have distinct effects on both acyl chain disorder and average acyl chain packing and that the properties of highly unsaturated membranes are not equivalent to more saturated membranes at a higher temperature.

2.5. Interaction With Cholesterol

It is well established that cholesterol has a strong ordering effect on saturated phospho-lipid acyl chains in the fluid phase. This results in condensation of phospholipid mono-layers and a reduction in enthalpy of the main gel to liquid-crystalline phase transition. These effects are reduced as sn-2 acyl chain unsaturation is increased for phospholipids with a saturated sn-1 chain (Hernandez-Borrell et al., 1993; Smaby et al., 1997), although the proximity of the double bonds to the headgroup is as important as the number of double bonds (Stillwell et al., 1994).

For all symmetrically substituted, unsaturated PCs, the chain-ordering effect of cholesterol is greatly reduced when compared with the corresponding sn-1 saturated, sn-2 unsaturated PC, and the effect of cholesterol decreases as the level of unsaturation increases (Mitchell & Litman, 1998b). A few studies have examined the effects of cholesterol in bilayers consisting of dipolyunsaturated PCs. All of these studies demonstrate that even at concentrations above 30 mol%, cholesterol has very little effect on the acyl-chain-packing properties of dipolyunsaturated bilayers. In both di20:4 PC and di22:6 PC, cholesterol has almost no effect on the gel-liquid-crystalline phase transition (Kariel et al., 1991), causes minimal change in acyl chain packing (Mitchell & Litman, 1998b), and causes only a small increase in the monolayer elastic area compressibility modulus (Smaby et al., 1997). In 18:0,18:1 PC bilayers, 50 mol% cholesterol increases the elastic area expansion modulus by 600 dyn/cm, whereas in di20:4 PC, 50 mol% cholesterol increases this parameter by only 50 dyn/cm (Needham & Nunn, 1990). The best explanation of these observation comes from a deuterium NMR study, which showed that cholesterol is soluble in di20:4n6 PC only to 15 mol% and that the molecular organization of cholesterol in this bilayer is profoundly different from that observed in sn-1 saturated, sn-2 polyunsaturated bilayers (Brzustowicz et al., 1999).

In recent years, much evidence has accumulated for lateral membrane domains that differ in their relative cholesterol content (Schroeder et al., 1995). In addition, it has been proposed that high levels of sn-2 unsaturation may promote formation of microdomains, in which the saturated sn-1 chains preferentially interact with each other (Litman et al., 1991). Several studies of cholesterol in bilayers containing high levels of polyunsaturation have reported evidence of lateral domains, which are driven by the preference of cholesterol for saturated acyl chains over polyunsaturated acyl chains (Huster et al., 1998; Mitchell & Litman, 1998b; Polozova & Litman, 2000; Zerouga et al., 1995). The recent

Fig. 3. Schematic representation of lateral domains in a bilayer consisting of di16:0 PC (dark ovals), di22:6 PC(striated ovals), cholesterol (small, light ovals) in a 7 : 3 : 3 ratio and rhodopsin (large gray ovals) at a 100 : 1 ratio of PC : rhodopsin. Rhodopsin is in a cluster, highly enriched in 22:6n-3 acyl chains, whereas cholesterol is mainly associated with the saturated 16:0 acyl chains. The enrichment of di22:6 PC in the cluster around rhodopsin is enhanced about six times relative to the bulk concentration. The cluster extends about three layers around rhodopsin.

Fig. 3. Schematic representation of lateral domains in a bilayer consisting of di16:0 PC (dark ovals), di22:6 PC(striated ovals), cholesterol (small, light ovals) in a 7 : 3 : 3 ratio and rhodopsin (large gray ovals) at a 100 : 1 ratio of PC : rhodopsin. Rhodopsin is in a cluster, highly enriched in 22:6n-3 acyl chains, whereas cholesterol is mainly associated with the saturated 16:0 acyl chains. The enrichment of di22:6 PC in the cluster around rhodopsin is enhanced about six times relative to the bulk concentration. The cluster extends about three layers around rhodopsin.

work of Polozova and Litman (2000) is especially significant in terms of biological mechanisms, because it was found that in a mixed PC system composed of di16:0 PC and di22:6 PC, lateral domain behavior was observed only when both cholesterol and the integral membrane protein rhodopsin were included in the bilayer. A conceptual diagram of the proposed protein-containing microdomains is shown in Fig. 3. The observation that rhodopsin was essential for the formation of domains and showed a distinct preference for di22:6 PC indicates a mechanism whereby changes in either phospholipid acyl chain unsaturation or membrane cholesterol could control membrane domain formation and, thereby, integral membrane protein function.

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