Effects of Various Numbers and Positions of cis Double Bonds in the sn-2 Acyl Chain of Phosphatidylethanolamine on the Chain-melting Temperature*

Guoquan Wang, Shusen Li, Hainan Lin, Erich E. Brumbaugh, and Ching-hsien HuangDagger

From the Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In an attempt to investigate systematically the effects of various single and multiple cis carbon-carbon double bonds in the sn-2 acyl chains of natural phospholipids on membrane properties, we have de novo synthesized unsaturated C20 fatty acids comprised of single or multiple methylene-interrupted cis double bonds. Subsequently, 15 molecular species of phosphatidylethanolamine (PE) with sn-1 C20-saturated and sn-2 C20-unsaturated acyl chains were semi-synthesized by acylation of C20-lysophosphatidylcholine with unsaturated C20 fatty acids followed by phospholipase D-catalyzed base-exchange reaction in the presence of excess ethanolamine. The gel-to-liquid crystalline phase transitions of these 15 mixed-chain PE, in excess H2O, were investigated by high resolution differential scanning calorimetry. In addition, the energy-minimized structures of these sn-1 C20-saturated/sn-2 C20-unsaturated PE were simulated by molecular mechanics calculations. It is shown that the successive introduction of cis double bonds into the sn-2 acyl chain of C(20):C(20)PE can affect the gel-to-liquid crystalline phase transition temperature, Tm, of the lipid bilayer in some characteristic ways; moreover, the effect depends critically on the position of cis double bonds in the sn-2 acyl chain. Specifically, we have constructed a novel Tm diagram for the 15 species of unsaturated PE, from which the effects of the number and the position of cis double bonds on Tm can be examined simultaneously in a simple, direct, and unifying manner. Interestingly, the characteristic Tm profiles exhibited by different series of mixed-chain PE with increasing degree of unsaturation can be interpreted in terms of structural changes associated with acyl chain unsaturation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Most naturally occurring diacyl phospholipids in eukaryotic cell membranes are of a mixed acyl chain variety, meaning that the fatty acids esterified at the sn-1 and sn-2 positions of the glycerol backbone are originated primarily in vivo from saturated and unsaturated fatty acyl-CoA, respectively. Since the chemical composition of fatty acids can vary greatly in terms of the acyl chain length, the degree of unsaturation, and the position of cis carbon-carbon double bonds (Delta -bonds),1 membrane phospholipids are structurally an extremely diverse group of amphipathic molecules. In a given type of cell, membrane phospholipids may amount to several hundreds of distinctive chemical species. Despite the bewildering diversity, the basic motif of most unsaturated fatty acyl chains is surprisingly simple, viz. in the sn-2 acyl chain, the cis carbon-carbon double bonds are invariably separated by a three-carbon unit comprised of a methylene group (-CH2-) sandwiched by two olefinic carbons. The biochemical significance of this regular methylene-interrupted interval is, however, not clear.

Although it has long been known that mixed-chain diacyl phospholipids in aqueous media can uniquely assemble into a two-dimensional sheet-like structure called the lipid bilayer, progress is nonetheless slow in understanding how variations in the chemical composition of fatty acyl chains affect the structure/property relationship of the lipid bilayer. This is, in part, due to the fact that up to the present time not a single x-ray crystal structure of mixed-chain phospholipid is available. A second reason is that many diacyl mixed-chain phospholipids containing single or multiple cis double bonds are rather difficult to synthesize (or semi-synthesize) in a typical biochemical or biophysical laboratory. Nevertheless, the pioneer work of Keough and co-workers (1-3) did provide interesting results showing how variations in the number of cis double bonds in the sn-2 acyl chain of phosphatidylcholine (PC) at certain fixed positions can affect the phase transition temperature (Tm) of the lipid bilayer. Specifically, Keough and associates (1-3) have shown by DSC that the introduction of a cis double bond into the sn-2 acyl chain of C(20):C(20)PC at carbon 11 from the carbonyl end, or C(11), gives rise to C(20):C(20:1Delta 11)PC, which has a considerably lower Tm relative to its saturated counterpart. The introduction of a second cis double bond into the sn-2 acyl chain at the methylene-interrupted position toward the methyl end yields C(20):C(20:2Delta 11,14)PC with a further reduction in Tm. Interestingly, the introduction of a third cis double bond at C(17) results in a small increase in Tm for C(20):C(20:3Delta 11,14,17)PC. This down and up trend in Tm has been confirmed calorimetrically by other groups (4, 5). One can immediately raise a relevant question as to whether this down and up Tm profile is a special or a general characteristic for acyl chain unsaturation. Phrased differently, what kind of Tm profile will be observed if the first Delta -bond is introduced at C(5) or C(17) in the sn-2 acyl chain followed by successive incorporations of Delta -bonds at regular methylene-interrupted intervals, proceeding toward the methyl or carbonyl end? One may further ask an even more important question: do we know how to interpret the observed Tm profile in terms of molecular structures of unsaturated phospholipids? In order to find answers to these fundamental questions, phospholipids with Delta -bond(s) at different positions along the sn-2 acyl chain need to be synthesized first, and the synthesized lipids should then be subjected to DSC studies. Furthermore, the structures of unsaturated lipids in the bilayer at T < Tm have to be estimated.

Recently, we have semi-synthesized a limited number of diacyl mixed-chain phosphatidylethanolamines (PE) with sn-1 C20-saturated and sn-2 C20-unsaturated acyl chains (6-9). Our calorimetric data showed that the Tm profile exhibited by a series of mixed-chain PE containing 1-3 cis Delta -bonds in the sn-2 C20-acyl chains at Delta 11-, Delta 11,14-, and Delta 11,14,17-positions, respectively, is parallel to that displayed by the corresponding mixed-chain PC observed earlier by Keough et al. (8). The influence of single and multiple Delta -bonds on the chain-melting behavior of PC and PE bilayers thus appears very similar. If we can delineate the common structural features governing the shapes of Tm profiles for various series of mixed-chain PE, the information obtained may provide an understanding of the structure/property relationships underlying most other naturally occurring phospholipids. With this in mind, in the present study we have extended the list of synthesized mixed-chain PEs containing sn-1 C20-saturated and sn-2 C20-unsaturated acyl chains to a total of 15 different species. The phase transition behavior of these mixed-chain PE, in excess H2O, has also been investigated by high resolution DSC. Based on the calorimetric data, a novel Tm diagram is generated for the first time. In this diagram, Tm values of 9 series of mixed-chain PE each containing three or more lipids are systematically arranged. The shapes of Tm profiles displayed by various lipids in all nine series can be seen simultaneously in plots derived from the Tm diagram. Furthermore, in this study we have used the computer-based molecular mechanics (MM) calculations to simulate the energy-minimum structures of these mixed-chain PEs. The characteristic Tm profile obtained with lipids in each series of mixed-chain PE can be interpreted in terms of structural changes of the sn-2 acyl chain of the lipid resulting from acyl chain unsaturation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- With the exception of six species of unsaturated C20 fatty acids that were synthesized in this laboratory as described in the next paragraph, all other C20-unsaturated fatty acids including arachidonic acid were obtained from Sigma. Lysophosphatidylcholine with a C20-acyl chain was purchased from Avanti Polar Lipids (Alabaster, AL). Phospholipase D, type I from cabbage, was obtained from Sigma. Chemicals used for the fatty acid synthesis were supplied by Aldrich. All routine reagents and organic solvents were of reagent and spectroscopic grades, respectively, and they were obtained from various commercial sources.

Synthesis of Unsaturated C20 Fatty Acids and Semi-synthesis of Mixed-chain PE-- In the present investigation, six species of C20-unsaturated fatty acids were synthesized; they were cis-14-eicosenoic, cis-17-eicosenoic, cis,cis-5,8-eicosadienoic, cis,cis-8,11-eicosadienoic, cis,cis-14,17-eicosadienoic, and all-cis-8,11,14,17-eicosatetraenoic acids. For monoenoic acids, the synthesis was carried out based on the method of Holman and co-workers (10, 11). By using the synthesis of cis-14-eicosenoic acid as an example, this method can be briefly described as follows: the starting material is 1-bromoundecan-11-ol. After the hydroxyl group has been protected by 3,4-dihydro-2H-pyran, the primary alkyl derivative can interact with heptyne-1 in the presence of butyl lithium to yield omega -hydroxyl alkyne. Upon further reacting with CH2(CO2Et)2 in the presence of EtONa, the chain elongation step gives a product of appropriate total number of carbons, viz. the eicosa-14-ynoic acid. Finally, the triple bond of this C20-alkynoic acid is cis-hydrogenated using Lindlar catalyst to form cis-14-eicosenoic acid. The syntheses of various dienoic C20 fatty acids were accomplished by the established procedure published recently from this laboratory (9). For the synthesis of all-cis-8,11,14,17-eicosatetraenoic acid, the method of Osbond et al. (12) was employed, in which 1-bromoundeca-2,5,8-triyne and 8-nonyoic acid were coupled to form 8,11,14,17-eicosatetraynoic acid followed by cis-hydrogenation to form the final product of all-cis-tetraenoic acid. The purity of the synthesized mono- and dienoic acids was estimated by high pressure liquid chromatography.

Mixed-chain sn-1 C20-saturated/sn-2 C20-unsaturated PE was semi-synthesized from the corresponding PC by the base-exchange reaction in the presence of excessive amounts of ethanolamine hydrochloride, at pH 5.6, using phospholipase D according to the method of Comfurius and Zwaal (14) as described in detail elsewhere (6, 8). The semi-synthesis of mixed-chain PC involved the acylation of C(20)-lysophosphatidylcholine with C20 fatty acid in the presence of catalyst 4-pyrrolidinopyridine using the established procedure reported previously (15). In semi-synthesizing mixed-chain PE, all chemical and enzymatic reactions were carried out strictly under N2 to avoid possible lipid oxidation. All lipids were purified by column chromatography on silica gel 60, with which a mixture of CHCl3, CH3OH, 5% NH4OH, 175:35:4 (v/v/v) was used as the eluant. Only a single spot was observed for each of the PE synthesized, after about 1 µmol per sample was loaded on the thin layer plate and developed in CHCl3, CH3OH, 5% NH4OH (65:30:5). Prior to use, the lipid powder obtained from lyophilization of the lipid/benzene solution was kept at -20 °C.

High Resolution DSC Measurements-- The lipid samples used for DSC experiments were prepared according to our previously reported protocols (9). Specifically, the lyophilized lipid power was dispersed in cold aqueous buffer solution containing 50 mM NaCl, 0.25 mM diethylenetriaminepentaacetic acid, 5 mM phosphate buffer, pH 7.4, and 0.02 mg/ml NaN3. All DSC experiments were performed on a MicroCal MC-2 calorimeter with a DA-2 digital interface and data acquisition utility for automatic collection (Microcal, Northampton, MA). In these DSC runs, a constant heating scan rate of 15 °C/h was used; lipid samples were scanned a minimum of three times with at least 60-90 min of equilibration at low temperatures between scans. As in our previous studies (6-9), the phase transition temperature and the transition enthalpy were obtained from the second DSC heating curve. Specifically, the gel-to-liquid crystalline phase transition temperature, Tm, corresponds to the peak position with maximal peak height, and the transition enthalpy, Delta H, can be determined from the area under the transition peak and the lipid concentration using the software provided by Microcal. In general, the Tm value obtained at the transition with maximal peak height from the second DSC heating run was reproduced at ± 0.1 °C for each lipid sample. The Delta H value, however, has a considerable higher error owing to the uncertainty in deciding the onset and completion temperatures of the transition curve. The relative errors may amount to 20% for very broad transition curves as exhibited by some polyunsaturated lipids.

Molecular Mechanics (MM) Calculations-- All molecular mechanics (MM) force field calculations were carried out using an IBM RS/6000 computer workstation. The software MM3 (version 92) for MM calculations was supplied by Quantum Chemistry Program Exchange, Chemistry Department, Indiana University, Bloomington, IN. The MM3 computation began with the input of the estimated atomic coordinates for mixed-chain PE followed by systematic adjustment of the structural parameters by repeated automatic cycles of the Newton-Raphson minimization technique (16). These cycles of self-adjusted computation came to a halt as the steric energy reached the minimum. The structural data resulting from the MM3 computation were stored. Subsequently, these data were transferred into a Pentium P5-200 platform equipped with HyperChem 4.0 software (HyperCube, Gainesville, FL), from which the three-dimensional graphic images of the energy-minimized lipid molecules can be visualized. Details of the procedure for obtaining the energy-minimized structure for each sn-1 saturated/sn-2 unsaturated phospholipid were described previously (17-19). It should be mentioned, however, that prior to stochastic search for the energy-minimized conformation of sn-1 saturated/sn-2 unsaturated PE, the atomic coordinates (e.g. torsion angles) of the initially crude structure for a given mixed-chain PE were estimated based on the single crystal structure of C(12):C(12)PE (20) and the energy-minimized unsaturated fatty acyl chain (18). Any additional methylene-interrupted cis double bonds for the sn-2 acyl chain were constructed by using s-Delta s+s+Delta s- (or s+Delta s-s-Delta s+) as the added sequence (18), where s± refers to skew (±) conformations with torsion angles of about ± 110o and Delta  denotes cis double bond with torsion angle of about 0o. These initial coordinates of the crude structural model were used as a set of initial input data in MM3 computations.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Phase Transition Behavior of Lipid Bilayers Composed of PE with sn-1 Saturated C20 and sn-2 Unsaturated omega 3(or omega 6)C20 Acyl Chains-- Fig. 1 shows the second DSC heating curves for aqueous dispersions prepared individually from a saturated identical chain C(20):C(20)PE and its five unsaturated omega 3 derivatives. These unsaturated omega 3PEs contain various numbers of cis carbon-carbon double bonds in the sn-2 acyl chain of the lipid, with the position of the commonly shared double bond being 3 carbons from the methyl end (the omega 3-position) or 17 carbons from the carbonyl end (the Delta 17-position). The aqueous dispersion of C(20):C(20)PE is characterized by a single, sharp endothermic transition peaked at 82.5 °C; this peak temperature corresponds to the gel-to-liquid crystalline (or chain-melting) phase transition temperature, Tm, as reported previously (21). After the first cis carbon-carbon double bond is introduced into the sn-2 acyl chain of C(20):C(20)PE at the omega 3 (or Delta 17)-position, the resulting C (20):C(20:1Delta 17)PE also displays a single, sharp endothermic transition; however, its Tm decreases by 15.7 °C, relative to its saturated counterpart, from 82.5 to 66.8 °C (Fig. 1). This single endothermic transition can also be attributed to the gel-to-liquid crystalline phase transition. Furthermore, as a cis double bond is introduced successively at a regular methylene-interrupted interval toward the carbonyl end, various polyunsaturated PEs with increasing numbers of double bonds are formed. These polyunsaturated PEs, in excess water, display calorimetrically broad gel-to-liquid crystalline phase transitions each with a characteristic Tm (Fig. 1). Interestingly, in the plot of Tm versus the number of double bonds as illustrated in the inset of Fig. 1, the Tm value is observed to decrease nonlinearly with a stepwise increase in the number of cis carbon-carbon double bonds in the sn-2 acyl chain. In particular, the Tm increment is diminished substantially between C(20):C(20:3Delta 11,14,17)PE and C(20):C(20:5Delta 5,8,11,14,17)PE. This nonlinear decrease in Tm reflects that the successive increase in the degree of acyl chain unsaturation has a nonadditive Tm lowering effect. Nevertheless, all experimental Tm values fit reasonably well by a least squares binomial curve with a correlation coefficient of 0.9836 (the inset of Fig. 1). Values of Tm, Delta H, and other thermodynamic parameters associated with the gel-to-liquid crystalline (or chain-melting) phase transition for all five species of unsaturated omega 3 derivatives of C(20):C(20)PE are summarized in Table I.


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Fig. 1.   The representative second DSC heating curves for aqueous dispersions prepared individually from C(20):C(20)PE and its five unsaturated omega 3 derivatives. These unsaturated omega 3PEs contain 1-5 cis Delta -bonds in the sn-2 acyl chain of the lipid, with the position of the commonly shared double bond being 3 carbons from the methyl end (the omega 3- or Delta 17-position). The abbreviated name for each unsaturated lipid species is indicated under each transition curve, and above the transition curve, the value of Tm obtained with each of the six lipid dispersions is also indicated. Furthermore, the Tm value obtained with each individual omega 3PE is plotted in the inset against the number of cis Delta -bonds in that lipid species.

                              
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Table I
The thermodynamic parameters associated with the chain-melting phase transitions of C(20):C(20)PE and its unsaturated omega 3 and omega 6 derivatives

In Fig. 2, the second DSC heating curves for aqueous dispersions prepared from four unsaturated omega 6PEs and their common parent compound, C(20):C(20)PE, are illustrated. It is evident that all these DSC curves display single endothermic transitions each with a distinct Tm value. Moreover, a nonlinearly decreased Tm curve in the plot of Tm versus the number of cis carbon-carbon double bonds is observed (the inset of Fig. 2). In particular, the lipid species with an sn-2 arachidonyl (or all-cis-5,8,11,14-eicosatetraenoyl) chain has the smallest Tm value of 6.6 °C. It should be mentioned that the phase transition behavior of C(20):C(20)PE and some of its omega 6-unsaturated derivatives has been reported recently from this laboratory (8). However, the recently reported DSC thermograms were incomplete; for instance, the one displayed by the aqueous dispersion of C(20):C(20:1Delta 14)PE was missing. In contrast, Fig. 2 comprises all DSC thermograms for PEs with sn-1 C20-saturated/sn-2 C20-omega 6-unsaturated acyl chains. In Table I, the values of Tm, Delta H, and other thermodynamic parameters associated with the phase transition of C(20):C(20)PE and its four omega 6-unsaturated derivatives are summarized.


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Fig. 2.   The representative second DSC heating curves for aqueous dispersions prepared individually from a saturated identical chain C(20):C(20)PE and its four unsaturated omega 6 derivatives. These unsaturated omega 6PEs contain 1-4 cis Delta -bonds in the sn-2 acyl chain of the lipid, with the position of the commonly shared double bond being 6 carbons from the methyl end (the omega 6- or Delta 14-position). The phase transition temperature, Tm, for each lipid dispersion is plotted in the inset against the number of cis Delta -bonds in the sn-2 acyl chain of the corresponding omega 6PE.

As described under "Experimental Procedures," the monounsaturated C(20):C(20:1Delta 14)PE was semi-synthesized by acylation of C(20)-lyso-PC with cis-14-eicosenoic acid followed by base-exchange reaction in the presence of excessive ethanolamine. Furthermore, the monoenoic omega 6 fatty acid (cis-14-eicosenoic acid) was also synthesized in this laboratory. The single and symmetric endothermic transition with Tm = 47.4 °C and Delta T1/2 = 0.9 °C exhibited by the C(20):C(20:1Delta 14)PE dispersion as seen in Fig. 2 can be taken as strong evidence indicating that the monounsaturated omega 6PE and the monoenoic omega 6 fatty acid synthesized in this laboratory have high degrees of isomeric and chemical purities. Furthermore, the single phase transition at 16.4 °C exhibited by the aqueous dispersion of C(20):C(20:4Delta 8,11,14,17)PE is seen in Fig. 1 to be symmetrical with a Delta T1/2 of 2.4 °C, reflecting again the high degree of purity of our synthesized all-cis-8,11,14,17-eicosatetraenoic acid as well as the synthesized C(20):C(20:4Delta 8,11,14,17)PE.

The Tm Diagram for PE with sn-1 C20-saturated/sn-2 C20-unsaturated Acyl Chains-- In recent years, the phase transition behavior of six lipid species of C(20):C(20)PE derivatives with omega 9, omega 12, and omega 15 fatty acids esterified at the sn-2 position of the glycerol backbone has been characterized by high resolution DSC in this laboratory (6-9). When the Tm values from these six PEs and those obtained with omega 3 and omega 6 PEs shown in Figs. 1 and 2 are codified, a general Tm diagram for sn-1 C20-saturated/sn-2 C20-unsaturated PE can be generated for the first time (Fig. 3). Specifically, the Tm diagram shown in Fig. 3 has the shape of a right-angled triangle, comprising 15 species of sn-1 C20-saturated/sn-2 C20-unsaturated PE. These lipids are arranged into 5 levels depending on the position of the omega -carbon, where the omega -carbon is defined as the first olefinic carbon atom in the lipid's sn-2 acyl chain when counting from the methyl end of the chain. The five parallel levels of unsaturated lipids are layered from top to bottom according to the following order: omega 15PE, omega 12PE, omega 9PE, omega 6PE, and omega 3PE. Furthermore, the unique Tm value of any given sn-1 C20-saturated/sn-2 C20-unsaturated PE is shown under the abbreviated name of the given lipid species in Fig. 3. Vertically, each column in the Tm diagram also represents a series of unsaturated PEs, which share a common Delta n-bond. Hence, each series is designated as the Delta nPE series, where the superscript n denotes the position of the common cis carbon-carbon double bond (Delta -bond) in the sn-2 acyl chain when counting from the carbonyl end. In this case, the carbonyl carbon is designated as the first carbon, or C(1), of the acyl chain. Next, we shall see that with this Tm diagram, the effects of acyl chain mono- and polyunsaturation on the chain-melting behavior of lipid bilayers can be examined directly in a unifying manner.


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Fig. 3.   The Tm diagram for sn-1 C20-saturated/sn-2 C20-unsaturated PE. Lipids in each row share a common omega -carbon, where the omega -carbon is defined as the first olefinic carbon atom in the sn-2 acyl chain of the lipid when counting from the chain terminal methyl end. The five parallel rows of unsaturated lipids from top to bottom are omega 15PE, omega 12PE, omega 9PE, omega 6PE, and omega 3PE series as indicated. Lipids in each column share a common Delta n-bond, and lipids in each column thus belong to a Delta nPE series, where the superscript n denotes the position of the common cis carbon-carbon double bond in the sn-2 acyl chain when counting from the carbonyl end. In this case, the carbonyl carbon is designated as the first carbon of the acyl chain. The five columns of unsaturated lipids from left to right are Delta 17PE, Delta 14PE, Delta 11PE, Delta 8PE, and Delta 5PE series, respectively, as indicated. All together, there are 15 molecular species of unsaturated mixed-chain PE in this Tm diagram, and their Tm values are given under the abbreviated names of the corresponding mixed-chain PE.

For lipid species aligned horizontally along each row in the Tm diagram (Fig. 3), the added cis carbon-carbon double bond is introduced at a regular methylene-interrupted interval, proceeding toward the carbonyl end of the sn-2 acyl chain. In addition, this Tm diagram shows another common feature exhibited by each series of the omega (3-12)PE as follows. The Tm decreases continuously but nonlinearly with a stepwise increase in the number of cis double bonds. Consequently, we can arrive at a general conclusion that the gel-to-liquid crystalline phase transition behavior of PE bilayers is influenced markedly in a systematic way by the number of cis carbon-carbon double bonds present in the sn-2 acyl chain of the PE.

The lipid species aligned vertically along each column in the Tm diagram also show a growing number of cis double bonds in the sn-2 acyl chain of the lipid (Fig. 3). However, the methylene-interrupted cis double bond is added on the methyl side of the existing double bond. Interestingly, the Tm values exhibited by the Delta 8-PE series of C(20):C(20:1Delta 8)PE, C(20):C(20:2Delta 8,11)PE, C(20):C(20:3Delta 8,11,14)PE, and C(20):C(20:4Delta 8,11,14,17)PE and the Delta 11-PE series of C(20):C(20:1Delta 11PE, C(20):C(20:2Delta 11,14)PE, and C(20):C(20:3Delta 11,14,17)PE show down and up trends, which can be viewed as an exception of the continuously downward trend of Tm normally observed with most lipid series that contain a stepwise increase in the number of cis double bonds. A similar down and up Tm trend has been detected earlier for a Delta 11-PC series of C(20):C(20:1Delta 11PC, C(20):C(20:2Delta 11,14)PC, and C(20):C(20:3Delta 11,14,17)PC by Keough et al. (1, 3).

The Tm diagram shown in Fig. 3 can also be viewed diagonally. For the monoenoic series of PE, the lipid with the cis double bond located in the middle of the sn-2 acyl chain, C(20):C(20:Delta 11)PE, has the lowest Tm value of 43.3 °C; moreover, the Tm value increases as the single cis bond migrates from the middle toward the carbonyl or methyl end (Fig. 3), thereby resulting in a roughly V-shaped Tm profile. Similar Tm profiles can also be recognized for dienoic and trienoic series of PE as seen diagonally in the Tm diagram (Fig. 3). Based on these diagonal terms, one can conclude that for lipids with a fixed number of cis double bonds, the position of the double bonds along the acyl chain of the lipid can notably affect the chain-melting behavior. It should be mentioned that various V-shaped Tm profiles for the monoenoic, dienoic, and trienoic series of PE shown in the plot of Tm versus the position of cis double bonds have been documented previously, albeit separately, in the literature (6, 8, 9). Uniquely, the Tm diagram illustrated in Fig. 3 shows simultaneously all these Tm profiles in a simple and unifying manner.

The most important conclusion that can be drawn from the Tm diagram illustrated in Fig. 3 is that the number and the position of cis double bonds in the sn-2 acyl chain of PE can characteristically influence the gel-to-liquid crystalline phase transition temperature of the PE bilayer. To recapitulate this important point further, all Tm values of C(20):C(20)PE derivatives shown in Fig. 3 are plotted three-dimensionally against the number and the position of cis double bonds. Specifically, in the plot of Fig. 4A, omega (n)-carbon represents the first olefinic carbon at the position of n from the methyl end that is shared by all lipids in a series of unsaturated PE. It is evident that all Tm values within each series of the omega (3-12)PE as depicted in Fig. 4A exhibit a common feature, a continuously downward shift in Tm as the degree of chain unsaturation increases successively. In addition, roughly V-shaped Tm profiles are shared by mono-, di-, and trienoic series of PE as the position of cis double bonds migrates from the omega 3-carbon to the omega 15-, omega 12-, and omega 9-carbons, respectively, in the sn-2 acyl chain. In Fig. 4B, the position of the commonly shared cis bond is represented by the Delta n-carbon, where the superscript n denotes the position of the first olefinic carbon in the sn-2 acyl chain when counting from the carbonyl end. Discernibly, for lipids in the Delta 8-PE or Delta 11-PE series the variation of Tm is characterized by a down and up trend.


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Fig. 4.   Tm values of unsaturated mixed-chain PE shown in Fig. 3 are plotted three-dimensionally against the number and the position of cis double bonds. In the plot of A, omega (n)-carbon represents the first olefinic carbon at the position of n from the methyl end. B, the position of the commonly shared cis Delta -bonds is Delta n-carbon, where the superscript n denotes the position of the first olefinic carbon in the sn-2 acyl chain when counting from the carbonyl end.

The Energy Minimized Structures of sn-1 C20-saturated/sn-2 C20-unsaturated PE as Determined by Molecular Mechanics Simulations-- X-ray crystallographic structures of diacyl phospholipids with sn-1 saturated and sn-2 unsaturated chains have not been determined at the present time. However, based on the atomic coordinates of the single crystal structures of saturated diacyl phospholipids and unsaturated fatty acids, the energy-minimized structures of some unsaturated phospholipids in the crystalline state bilayer have been simulated by molecular mechanics (MM) calculations (18, 19). This MM approach is employed in the present study to construct the minimum energy structures for various PEs with sn-1 C20-saturated/sn-2 C20-unsaturated acyl chains.

In Fig. 5, the energy-minimized structures of six lipid species in the omega 3PE series are presented using space-filling and wire models. These structures can be taken to approximate the monomeric lipids packed in the crystalline bilayer. Here, the head groups of all six lipid molecules are aligned identically. Furthermore, the zigzag plane of the all-trans sn-1 acyl chain of each individual lipid species is seen in the wire model to lie perpendicularly to the paper plane, whereas the sn-2 acyl chain is seen in the space-filling model to project in front of the sn-1 acyl chain. For C(20):C(20)PE, the segment of the sn-2 acyl chain running approximately in parallel with the all-trans sn-1 acyl chain extends from C(3) to C(20) with 17 C-C bond lengths. However, the all-trans segment (ATS) of the sn-2 acyl chain is assumed to extend from C(3) to C(19) with 17 methylene units (Fig. 5). This assumption is based on the notion that the chain terminal CH2-CH3 bond is usually disordered at T < Tm, particularly for lipids packed in the gel state bilayer. In the case of monounsaturated C(20):C(20:1Delta 17)PE, the sequence of the Delta 17-containing kink in the sn-2 acyl chain is s-Delta s-, where s- and Delta  are skew(-) and cis double bonds, respectively. By MM calculations, a set of optimal torsion angles for this s-Delta s- sequence is determined to be (-109°, -1.1°, and -120°) as indicated in Fig. 5. Consequently, the sn-2 acyl chain of C(20):C(20:1Delta 17)PE has a crankshaft-like topology in which a long and a short chain segments separated by the Delta 17-bond can be identified. The long chain segment extends from C(3) to C(17) with 14 C-C bond lengths, and the short chain segment including the methyl end is only 2 C-C bond lengths long. In this communication, we define the all-trans segment in the long chain segment of the kinked sn-2 acyl chain as ATS. In the case of C(20):C(20:1Delta 17)PE, the ATS has 14 consecutive methylene units as indicated in Fig. 5. It should be noted that ATS is one C-C bond length shorter than that of the long chain segment due to the fact that the C-C single bond preceding the Delta 17-bond has a skew(-) conformation with an optimal torsion angle of -109°. For polyunsaturated omega 3PEs, the energetically most favorable structures obtained with MM calculations are also included in Fig. 5. Here, the sn-2 acyl chains are seen to adopt roughly an overall kinked motif. In particular, the optimal torsion angles for the sequences containing s± and Delta  bonds (the kink sequences) are given under the two molecular models of each energy-minimum structure. It should be noted from Fig. 5 that the short chain segment succeeding the Delta 17-bond is identical in length for all unsaturated omega 3PEs; in contrast, the length of ATS preceding the kink sequence in the sn-2 acyl chain decreases progressively with increasing numbers of Delta -bonds.


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Fig. 5.   The energy-minimized structures of identical-chain C(20):C(20)PE and its five unsaturated omega 3 derivatives as shown molecular graphically by space-filling and wire models. These unsaturated omega 3PEs contain 1-5 cis Delta -bonds. The ATS shown in the wire model denote the all-trans segment of the hydrocarbon chain in the sn-2 acyl chain. The kink sequence in the sn-2 unsaturated acyl chain and a set of optimal torsion angles associated with the kink sequence are presented below each unsaturated lipid species. It should be noted that the length of ATS is shortened progressively as the new methylene-interrupted cis-double bond is added successively on the carbonyl side of the Delta 17-bond in the sn-2 acyl chain of C(20):C(20:1Delta 17)PE.

The energy-minimized structures of the five lipids listed in the right column in the Tm diagram (Fig. 3) have also been determined by MM calculations using the MM3 program (data not shown). Like lipids in the omega 3PE series shown in Fig. 5, lipids in this Delta 5PE series contain up to 5 methylene-interrupted cis carbon-carbon double bonds in the sn-2 acyl chain. The chemical and molecular structures of these lipids, however, differ from those in the omega 3PE series. Specifically, the cis carbon-carbon double bond of the monounsaturated lipid lies in between C(5) and C(6) near the carbonyl end, and it is designated as the Delta 5-bond. The polyunsaturated lipids have their methylene-interrupted double bonds added on the methyl side of the Delta 5-bond. As a result, the short chain segment of the kinked sn-2 acyl chain is invariable in length, 2 C-C bond lengths, extending from C(3) to C(5) for all lipids in this series. In contrast, the length of ATS is shortened progressively as the new cis double bond is added successively into the sn-2 acyl chain. This decreasing trend in ATS is, in essence, identical to that observed in Fig. 5 for the omega 3 series of C(20):C(20)PE.

The minimum-energy structures of C(20):C(20:1Delta 11)PE, C(20):C(20:2Delta 11,14)PE, and (20):C(20:3Delta 11,14,17)PE in the Delta 11PE series are illustrated in Fig. 6 by space-filling and wire models. For C(20):C(20:1Delta 11)PE, the Delta 11-double bond is seen to be in the middle of the sn-2 acyl chain, and the additional Delta -bonds are on the methyl side of the Delta 11-bond. Unlike the variable length of ATS shown in Fig. 5, all three unsaturated lipid species in this series of Delta 11PE share a constant chain length of ATS extending from C(3) to C(10) in the sn-2 acyl chain.


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Fig. 6.   Molecular graphics representations of the energy-minimized structures of C(20):C(20:1Delta 11)PE, C(20):C(20:2Delta 11,14)PE, and (20):C(20:3Delta 11,14,17)PE in the Delta 11PE series as depicted by space-filling and wire models. It should be noted that ATS in each lipid species is located in the upper segment of the kinked sn-2 acyl chain, and the added Delta -bond is introduced on the methyl side of the existing Delta -bond. Here, the ATS is defined as the all-trans segment of consecutive -CH2- groups located in the long segment of the kinked sn-2 acyl chain. The kink sequence and associated torsion angles for each unsaturated acyl chain are presented under the energy-minimized structure of the corresponding lipid. These torsion angles were obtained with the MM3 program as described under "Experimental Procedures."

In Fig. 7, the energy-minimized structures of lipids in the Delta 8PE series are illustrated using space-filling and wire models. These four; not five lipid species share a common segment of 5 consecutive methylene units, extending from C(3) to C(7), in the sn-2 acyl chain. For C(20):C(20:2Delta 8,11)PE, there is a segment of 7 consecutive methylene units in the sn-2 acyl chain located between the olefinic carbon of C(12) and the methyl terminus; this long linear segment extending from C(13) to C(19) is, by definition, the ATS. In the case of C(20):C(20:3Delta 8,11,14)PE, a segment of 4 consecutive methylene units lies between C(15) and C(20) in the sn-2 acyl chain. The length of this segment is 1 methylene unit shorter than that of the linear segment near the interface extending from C(3) to C(7). Consequently, the longer linear segment near the interface is designated as the ATS for C(20):C(20:3Delta 8,11,14)PE. This figure thus serves as an example to demonstrate that for certain series of lipids the ATS may switch its location along the sn-2 acyl chain as each additional Delta -bond is progressively introduced into the sn-2 acyl chain of the lipid.


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Fig. 7.   The energy-minimized structures of lipids in the Delta 8PE series as represented by space-filling and wire models. Initially, the ATS is located in the lower segment of the unsaturated sn-2 acyl chain. In accompanying the C(20):C(20:2Delta 8,11)PE right-arrow (20):C(20:3Delta 8,11,14)PE conversion, the ATS is seen to shift from the lower segment to the upper segment. Here, ATS is defined as the all-trans segment of consecutive -CH2- groups located in the long segment of the kinked sn-2 acyl chain. The torsion angles of the kink sequence in each lipid species are given under the two structural models of the appropriate lipid molecule.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Prior to the discussion of the chain-melting phase transitions exhibited by aqueous lipid dispersions prepared individually from C(20):C(20)PE and its unsaturated omega 3 derivatives, it is appropriate to first comment on the simulated structures of these omega 3PEs as shown in Fig. 5. To a first approximation, these structures correspond to the optimal and static structures of PE molecules packed in the crystalline-state bilayer at T < Tm. Unlike molecular dynamics simulations, the MM-simulated structure does not explicitly provide information about the dynamic nature of lipid molecule. For instance, the sn-2 acyl chains of all unsaturated omega 3PEs share a common chain terminal segment of C(16)-C(17)=C(18)-C(19)-C(20), in which the C-C single bonds are all rotationally highly dynamic at T < Tm. Hence, this disordered methyl-terminal segment does not undergo the thermally induced trans right-arrow gauche isomerizations at Tm. The dynamic nature of this short terminal segment is, however, not revealed by the MM-simulated structure. On the other hand, as the number of Delta -bonds in this series of omega 3PE increases stepwise from 0 to 5, the length of ATS in this series of PE is shortened systematically by a methylene-interrupted interval. These static structural features, which will be used to correlate with the Tm in the rest of the "Discussion," are clearly indicated in the MM-simulated structures as depicted in Fig. 5.

When a cis carbon-carbon double bond (Delta ) is introduced into a long hydrocarbon chain, the six atoms in the immediate vicinity of the Delta -bond, C-CH=CH-C, are coplanar. Although the rotational flexibility of the Delta -bond in the six-atom unit is highly restricted at physiological temperatures, paradoxically the C-C single bond preceding or succeeding the Delta -bond is rotationally highly flexible, in terms of torsion-angle fluctuations, even at very low temperature of -10 °C (17). Hence, when a Delta -bond is introduced into the sn-2 acyl chain of C(20):C(20)PE near either the carbonyl end at the Delta 5-position or the methyl end at the omega 3-position, the short chain segment of the kinked sn-2 acyl chain in the gel-state bilayer at T < Tm can be reasonably assumed to be highly disordered, and hence it contains virtually no trans rotamers. We believe that this assumption is justified by its utility in the following discussion.

Fundamentally, the thermally induced gel-to-liquid crystalline phase transition of the lipid bilayer occurring at Tm involves principally the trans right-arrow gauche rotational isomerizations of methylene groups about C-C single bonds along the acyl chains of the lipid (22). Since the short segment of the kinked sn-2 acyl chain is assumed highly disordered in the gel-state bilayer at T < Tm, it thus makes no contributions to the chain disordering process at Tm. However, consecutive methylene groups in both the ATS of the sn-2 acyl chain and the all-trans sn-1 acyl chain can be induced thermally to undergo the disordering process of trans right-arrow gauche isomerizations. When we compare the thermodynamic parameters (Tm, Delta H, and Delta S) associated with the chain-melting phase transition for unsaturated lipids in the omega 3PE series at T < Tm, we mention primarily the length of ATS. This is due to the fact that in the gel-state bilayer an identical length of the all-trans sn-1 C20-acyl chain exists in all lipids in the omega 3PE series. Remember that the C-C double bond is rotationally highly restricted. We, therefore, also take the rigidity of multiple Delta -bonds into consideration. Specifically, we suggest that the C-C double bond exerts its effect on the chain-melting phase transition when the Delta -bond in the sn-2 C20-acyl chain reaches the number of three.

The changes in the chain length of ATS and the number of Delta -bonds in the sn-2 acyl chain of the lipid can explain qualitatively the Delta H trend observed with lipids in the omega 3PE series as shown in Table I. Here, the Delta H values are seen to decrease initially with increasing number of Delta -bonds. In particular, the Delta H value is at a minimum for C(20):C(20:2Delta 14,17)PE with a sn-2 dienoyl chain; thereafter, Delta H values are virtually independent of the number of Delta -bonds. The transition enthalpy associated with the chain-melting transition of the bilayer is Delta H = Hlc - Hgel, where H is the enthalpy of the lipid bilayer and the subscripts lc and gel denote the liquid-crystalline and gel phases of the lipid bilayer, respectively. It is well known that saturated PE and its sn-1 saturated/sn-2 unsaturated derivatives are highly dynamic and disordered in lipid bilayers in the liquid-crystalline state; hence, the lateral chain-chain contact interactions are minimal among lipids in these liquid-crystalline bilayers. We can thus assume that Hlc is virtually identical for lipids with 0-5 Delta -bonds. As a result, the Delta H trend exhibited by lipids in the omega 3PE series can, to a first approximation, be related to the Hgel values for these lipids. For unsaturated lipids packed in the ordered gel-state bilayer, the lateral chain-chain van der Waals attractive interactions can be directly related to the length of ATS; moreover, these interactions are also modulated by the dynamic state of the ATS. As the first two cis C-C double bonds are introduced into the sn-2 acyl chain of C(20):C(20)PE at the Delta 17- and Delta 14,17-positions, the length of ATS decreases progressively (Fig. 5). In addition, the ATS as a whole also becomes more dynamic due to the high flexibility of the C-C single bonds adjacent to methylene-interrupted cis double bonds. Consequently, the lateral van der Waals attractive chain-chain interactions are weakened by the initial acyl chain unsaturation. The marked reduction in Delta H can thus be interpreted as follows: increasing up to two Delta -bonds there is a steady increase in Hgel as a result of progressive weakening of the overall chain-chain contact interactions. Beyond two Delta -bonds, the rigid multiple methylene-interrupted Delta -bonds are assumed to act as a structural unit in the sn-2 acyl chain which can facilitate the favorable lateral chain-chain contact interaction, thus resulting in a decrease in Hgel. This enhanced contact interaction evidently is enough to compensate the opposing effect of ATS resulting from incorporation of additional Delta -bonds. Consequently, for lipids with 3-5 Delta -bonds in the omega 3PE series, their Hgel and hence Delta H values are nearly identical.

Similarly, the transition entropy for the chain-melting phase transition can be expressed as Delta S = Slc - Sgel. For lipids in the omega 3PE series, the value of Slc can be assumed to decrease linearly with increasing number of Delta -bonds. This assumption is based on the fact that rotation of the C-C double bond is energetically prohibited; hence, a stepwise increase in the number of Delta -bonds corresponds to a progressive decrease in the randomness or entropy of the acyl chain of the lipid. In the gel-state bilayer, however, the effect of Delta -bonds on the Sgel value cannot be linear. Specifically, as the Delta -bond increases from 0 to 2, the rotational freedom of the lipid molecule as a whole or the Sgel value increases markedly due to the high flexibility of C-C single bonds adjacent to the Delta -bonds. Above 2, the Sgel decreases with increasing Delta -bonds as a result of the increased rigidity of multiple Delta -bonds. In particular, the maximal Sgel occurs at Delta -bonds of 2, where the C-C single bonds adjacent to Delta -bonds are highly flexible, whereas the overall rigidity of the two Delta -bonds is not sufficient to cause a substantial decrease in Sgel. Based on the proposed linear Slc curve and the nonlinear Sgel curve in the plot of S versus the number of Delta -bonds, a nonlinear Delta S curve with a minimum of transition entropy at Delta -bonds of 2 can be expected for lipids in the omega 3PE series. This expected Delta S trend is indeed qualitatively similar to the calculated Delta S values obtained with lipids in the omega 3PE series as shown in Table I.

For lipids in the omega 3PE series, the changes in Tm as a function of alterations in the number of Delta -bonds can now be considered. First, the following identity holds: Tm= Delta H/Delta S. Second, despite the scattering of the data the Delta H and Delta S both change in the same direction as the number of Delta -bonds varies. Specifically, the Delta H and Delta S both decrease markedly with increasing number of Delta -bonds up to 2; thereafter, they increase slightly and then remain nearly unchanged (Table I). Based on these relationships and the Tm profile observed in Fig. 1, we can conclude that the origin of Tm is largely enthalpic, not entropic. Hence, for each lipid in the omega 3PE series the main contribution to Tm is the overall lateral chain-chain attractive van der Walls interaction in the gel-state bilayer. However, in view of all lipids in the omega 3PE series, particularly those with 3-5 Delta -bonds, the relative Tm values must also be modulated differentially by entropic variations. As a result, the shape of the Tm profile varies somewhat from that of the Delta H profile. As discussed earlier, the chain-chain van der Walls attractive interaction depends largely on the length of ATS and the number of Delta -bonds in the sn-2 acyl chain. In addition, the small contribution of the entropic effect also relates to the ATS and Delta -bonds. Unfortunately, quantification of the relative contributions of ATS and Delta -bonds to Tm is not possible. Nevertheless, the relative magnitudes of Tm for lipids in the omega 3PE series can be correlated qualitatively with the variations in the structural parameters of ATS and Delta -bonds. We, therefore, propose that the nonlinear Tm profile seen in Fig. 1 (the inset) can be reasonably attributed to the net result of the following two opposing effects as follows: 1) the Tm lowering effect caused by the progressive shortening of ATS, and 2) the Tm elevating effect exerted by the increasing rigidity of 3-5 Delta -bonds. It is important to point out that the shortening of a three-carbon interval in the ATS has a more pronounced Tm lowering effect than the opposing effect of the added Delta -bonds for lipids in this series of omega 3PE. Consequently, the Tm profile seen in the inset of Fig. 1 is characterized by a decreasing, not an increasing, temperature mode. It is perhaps worth mentioning that the two opposing effects are caused paradoxically by the same structural change, viz. the increasing degree of acyl chain unsaturation in the sn-2 acyl chain.

We further postulate that the combined effects of the length of ATS and the multiple Delta -bonds on Tm discussed above are also operative in bilayer membranes composed of other series of PE with 3 or more Delta -bonds. The omega 6 and Delta 5 series of C(20):C(20)PE derivatives illustrated in the Tm diagram (Fig. 3) can serve as examples. In each of the two PE series, the monounsaturated lipid has its Delta -bond located near the methyl or carbonyl end, causing the sn-2 acyl chain kinked into two segments with unequal lengths. In particular, the long segment of the kinked acyl chain contains the highly ordered ATS. Furthermore, the incorporation of the additional Delta -bond always takes place in this long segment at a regular methylene-interrupted interval, resulting in a stepwise shortening of the ATS and hence a continuous decrease in Tm. However, the magnitude of the Tm reduction must be damped down somewhat due to an increasing number of Delta -bonds beyond three. Specifically, the multiple Delta -bonds in the sn-2 acyl chains of lipids in these two series of PE tend to promote higher Tm, but this Tm elevating effect is less than the Tm lowering effect exerted by the shortening of the ATS in the sn-2 acyl chain. On balance, the Tm values in the plot of Tm versus the number of Delta -bonds are expected to fall on nonlinearly decreasing curves for lipids from the omega 6-, or Delta 5-PE series. This expectation is indeed borne out by experimental data (Fig. 2, inset, and Fig. 3).

The monounsaturated C(20):C(20:1Delta 11)PE is a common lipid species shared by the omega 9PE and the Delta 11PE series as shown in the Tm diagram (Fig. 3). The topological feature of C(20):C(20:1Delta 11)PE is illustrated in Fig. 6I. In particular, the kinked cis-11-eicosenoyl chain consists of two roughly parallel segments, the upper and lower segments, jointed by a kink sequence of s-Delta s-g-. Here, the upper segment designates the chain segment closer to the bilayer/H2O interface, and the lower segment is assigned to the one containing the methyl group. In the kinked sn-2 cis-11-eicosenoyl chain of C(20):C(20:1Delta 11)PE, the ATS with 8 methylene units is located in the upper segment, extending from C(3) to C(10). The lower segment, however, has 7 methylene units extending from C(13) to C(19). If a new Delta -bond is incorporated successively into the cis-11-eicosenoyl chain at the regular methylene-interrupted interval, this process will yield two different series of unsaturated PE. Specifically, the omega 9PE and the Delta 11PE series are obtained if the incorporation of Delta -bonds takes place in the upper and lower segments of cis-11-eicosenoyl chain, respectively. Interestingly, the Tm profiles exhibited by these two series of unsaturated PE are distinctly different (Fig. 4, A and B), indicating that the position of the incorporated Delta -bond can markedly affect the chain-melting behavior. The shape of each of the two Tm profiles will be interpreted later in terms of the length and location of ATS and the rigidity of the multiple Delta -bonds.

In the gel-state bilayer, the polymethylene units in the upper chain segment of the acyl chain of the lipid are more ordered than those in the lower segment near the bilayer center. This is due largely to the fact that the upper segment is linked covalently at both ends, and the lower segment has a free and dynamic methyl terminus. The lateral chain-chain contact interactions are thus stronger for upper chain segments. Consequently, ATS in the upper chain segment can contribute somewhat more to the overall chain disordering process of trans right-arrow gauche isomerizations in comparison with the equivalent length of ATS located in the lower chain segment. Hence, when two lipids with the same length of ATS are compared, the one with ATS in the upper segment will have a higher Tm. Similarly, if a short segment of consecutive methylene units is in the upper portion of the sn-2 acyl chain and its length differs from that of ATS in the lower segment by only one or two -CH2- units, then this short segment can most likely undergo the thermally induced trans right-arrow gauche isomerizations. It thus contributes to the overall chain-melting process. With these basic concepts in mind, we can rationalize the characteristic Tm profile exhibited by lipids in the omega 9PE series.

The three lipids in the omega 9PE series, C(20):C(20:1Delta 11)PE, C(20):C(20:2Delta 8,11)PE, and C(20):C(20:3Delta 5,8,11)PE, share a common lower segment of 7 consecutive methylene units in their sn-2 acyl chains. As shown in Figs. 6I and 7II, the C(20):C(20:1Delta 11)PE right-arrow C(20):C(20:2Delta 8,11)PE conversion is accompanied by two distinctive structural changes as follows: the length of ATS is shortened by one -CH2- unit, and the position of ATS is shifted from the upper to the lower chain segment. Both changes can result in a lowering of Tm. Indeed a decrease of 13 °C in Tm is observed experimentally to accompany such a conversion (Fig. 3). The third member of the omega 9PE series is the trienoic C(20):C(20:3Delta 5,8,11)PE. Although the MM-simulated structure is not shown, this trienoic lipid contains an ATS that is identical to the corresponding ATS in the dienoic C(20):C(20:2Delta 8,11)PE. Despite the identical position and length of ATS, the Tm of C(20):C(20:3Delta 5,8,11)PE is observed calorimetrically to be smaller than that of C(20):C(20:2Delta 8,11)PE by 7.3 °C (Fig. 3). In Fig. 7II, cis, cis-8,11-eicosadienoyl chain is seen to have a short upper segment composed of 5 consecutive methylene units. As discussed in the preceding section, some of these 5 methylene units in the upper portion of the sn-2 acyl chain can make contributions to the overall chain disordering process of trans right-arrow gauche isomerizations. When the length of this relatively ordered segment of 5 methylene units is reduced by three carbon-carbon lengths as a result of the C(20):C(20:2Delta 8,11)PE right-arrow C(20):C(20:3Delta 5,8,11)PE conversion, its contribution to the overall chain-melting process is totally abolished, resulting in a lower Tm. However, all-cis-5,8,11-eicosatrienoyl chain has three Delta -bonds, which can elevate the Tm somewhat by retarding sterically the trans right-arrow gauche isomerizations of neighboring chains in bilayers. As a result, the Delta Tm between C(20):C(20:2Delta 8,11)PE and C(20):C(20:3Delta 5,8,11)PE is smaller than that between C(20):C(20:1Delta 11)PE and C(20):C(20:2Delta 8,11)PE.

Thus far, we have discussed four Tm profiles exhibited by lipids from omega 3-, omega 6-, omega 9-, and Delta 5PE series. All four Tm profiles show a similar pattern; the Tm decreases nonlinearly as the number of Delta -bonds in the sn-2 acyl chain of the lipid increases stepwise. Two other series of PE shown vertically in Fig. 3 are Delta 8- and Delta 11PE series; interestingly, the Tm profile displayed by lipids from each of the Delta 8- and Delta 11PE series is characterized by a down and up pattern as illustrated in Fig. 4B. Each of the down and up Tm profiles will be analyzed in the subsequent section; in particular, the structural features of those lipid species that give rise to higher Tm upon further unsaturation will be delineated. Let us begin with the Delta 11PE series.

The energy-minimum structures of the three lipids in the Delta 11PE series are illustrated in Fig. 6. One common feature shared by them is the identical length of the ATS located in the upper chain segment. As a result, the length of ATS alone cannot account for the variations of Tm observed with lipids in the Delta 11PE series; other structural features have to be considered. For C(20):C(20:1Delta 11)PE, the lower segment of the sn-2 acyl chain has 7 consecutive methylene units (Fig. 6I). Some of these -CH2- units, particularly those located far away from the chain methyl end, can make contributions to the chain-melting process of trans right-arrow gauche isomerizations, resulting in a higher Tm. The other two lipids in the same series are C(20):C(20:2Delta 11,14)PE and C(20):C(20:3Delta 11,14,17)PE, in which the short lower segments are relatively disordered at T < Tm. Moreover, their lengths are smaller than the corresponding short segment in C(20):C(20:1Delta 11)PE by 3 and 6 C-C methylene units, respectively, as shown in Fig. 6. Among the three lipids in the Delta 11PE series, C(20):C(20:1Delta 11)PE must, therefore, exhibit the highest Tm. Next, we continue the comparison between C(20):C(20:2Delta 11,14)PE and C(20):C(20:3Delta 11,14,17)PE. Structurally, the fundamental difference between them lies in the number of Delta -bonds. Earlier, we have postulated that the presence of three to five cis Delta -bonds in the sn-2 C20-acyl chain of PE can cause the bilayer to exhibit a higher Tm. As before, this Tm elevating effect is small in comparison with the opposing effect exerted by the reducing length of the chain segment during acyl chain unsaturation. This is, however, not applicable in the case of the C(20):C(20:2Delta 11,14)PE right-arrow C(20):C(20:3Delta 11,14,17)PE conversion. In particular, the number of consecutive methylene units present in the short lower segments of cis,cis-11,14-eicosadienoyl chain is 4 only (Fig. 6). This short lower segments in the gel-state bilayer is thus highly disordered, and it makes no contributions to the chain-melting process of trans right-arrow gauche isomerizations underlying the main phase transition at Tm. In going from C(20):C(20:2Delta 11,14)PE to C(20):C(20:3Delta 11,14,17)PE, a Delta -bond is introduced into this highly disordered segment of the sn-2 acyl chain at C(17), causing a further shortening of the lower segment. Since this segment is highly disordered prior to the conversion, a shortening of this segment by 3 -CH2- units upon unsaturation at C(17) will not affect appreciably the Tm. As a result, the opposing effect of the rigid triple Delta -bonds of C(20):C(20:3Delta 11,14,17)PE becomes dominant, and the relative magnitude of Tm between C(20):C(20:2Delta 11,14)PE and C(20):C(20:3Delta 11,14,17)PE thus becomes apparent. For the three lipids in the Delta 11PE series, their Tm can thus be rationalized to have the following decreasing order: C(20):C(20:1Delta 11)PE > C(20):C(20:3Delta 11,14,17)PE > C(20):C(20:2Delta 11,14)PE. This order will yield a down and up Tm curve in the plot of Tm versus the number of Delta -bonds.

The Delta 8PE series consists of the following four lipid species: C(20):C(20:1Delta 8)PE, C(20):C(20:2Delta 8,11)PE, C(20):C(20:3Delta 8,11,14)PE, and C(20):C(20:4Delta 8,11,14,17)PE. All of them share a common upper chain segment of 5 consecutive methylene units extending from C(3) to C(7) in the sn-2 acyl chain (Fig. 7). During the C(20):C(20:1Delta 8)PE right-arrow C(20):C(20:2Delta 8,11)PE conversion, the number of -CH2- units in the ATS of the sn-2 acyl chain decreases from 10 to 7, with the location of ATS being in the lower chain segment. A reduction in Tm is thus expected to accompany the conversion, and such a reduction has indeed been observed calorimetrically. The subsequent C(20):C(20:2Delta 8,11)PE right-arrow C(20):C(20:3Delta 8,11,14)PE conversion is characterized by a further shortening of three -CH2- units in the ATS of cis,cis-8,11,-eicosadienoyl chain, leading to a continuous decrease in Tm. In accompanying the C(20):C(20:2Delta 8,11)PE right-arrow C(20):C(20:3Delta 8,11,14)PE conversion, the ATS shifts its location from the lower to the upper chain segment (Fig. 7, II and III). This has an important implication, meaning that the subsequent incorporation of the fourth Delta -bond into all-cis-8,11,14-eicosatrienoyl chain at C(17) affects only the length of the lower chain segment. In particular, the length and position of ATS in the sn-2 acyl chain remain unchanged as shown in Fig. 7, IV. Consequently, the trienoic C(20):C(20:3Delta 8,11,14)PE and the tetraenoic C(20):C(20:4Delta 8,11,14,17)PE share a common length of ATS in their sn-2 acyl chains' upper segments. On the basis of the identical length of ATS alone, the Delta Tm associated with the C(20):C(20:3Delta 8,11,14)PE right-arrow C(20):C(20:4Delta 8,11,14,17)PE conversion would be zero. On the other hand, the number of Delta -bonds in the sn-2 acyl chain of C(20):C(20:4Delta 8,11,14,17)PE is higher than that in C(20):C(20:3Delta 8,11,14)PE. As proposed earlier, this additional Delta -bond may raise the Tm of C(20):C(20:4Delta 8,11,14,17)PE. Hence, on the basis of the Tm elevating effect of multiple Delta -bonds and the identical length of ATS, a positive Delta Tm can be expected to underlie the C(20):C(20:3Delta 8,11,14)PE right-arrow C(20):C(20:4Delta 8,11,14,17)PE conversion. Indeed, the expectation is borne out by DSC data presented in Fig. 3, in which the Tm value displayed by the aqueous dispersion of C(20):C(20:4Delta 8,11,14,17)PE is seen to be 0.8 °C higher than that of C(20):C(20:3Delta 8,11,14)PE.

Three series of mixed-chain PEs with fixed numbers of Delta -bonds can be seen along the diagonal lines in the Tm diagram (Fig. 3). The monoenoic PE series has a total number of five lipids. The di- and trienoic PE series consist of four and three lipids, respectively. In response to changes in the position of the double bond, the Tm values of lipids with a fixed number of double bonds give rise to a roughly V-shaped Tm profile (Fig. 4, A and B). Molecular interpretations of such a characteristic Tm profile have been given in detail elsewhere from this laboratory (9). Hence, we shall not discuss the V-shaped Tm profiles exhibited by mono-, di-, and trienoic PE.

To sum up, for a series of sn-1 saturated/sn-2 unsaturated mixed-chain PE containing different numbers of Delta -bonds, a continuously decreasing Tm profile is generally observed in the plot of Tm versus the number of Delta -bonds as exemplified by data shown in Figs. 1 or 2. However, there are exceptions. For instance, the C(20):C(20:2Delta 11,14)PE right-arrow C(20):C(20:3Delta 11,14,17)PE and the C(20):C(20:3Delta 8,11,14)PE right-arrow C(20):C(20:4Delta 8,11,14,17)PE conversions are coupled with increased Tm, and the Tm profiles observed with lipids in the Delta 8- and Delta 11PE series are thus characterized by a down and up trend. The mixed-chain PE that can, upon unsaturation, convert into a higher Tm species has the following structural characteristics: 1) the sn-2 acyl chain contains at least two methylene-interrupted cis Delta -bonds; 2) the number of consecutive methylene units in the upper chain segment is no fewer than that in the lower chain segment; 3) the Delta -bond to be further incorporated into the unsaturated sn-2 acyl chain must be added in the lower chain segment in the direction toward the methyl terminus. Furthermore, for mixed-chain PE with 20 carbon atoms in the sn-2 acyl chain, it is interesting to note that only omega 3 lipids such as C(20):C(20:3Delta 11,14,17)PE and C(20):C(20:4Delta 8,11,14,17)PE exhibit higher Tm values than their omega 6 precursors as shown calorimetrically in the present and previous studies (8, 23). The significance of the down and up Tm profile is that it means the polyunsaturated omega 3 lipid with its multiple Delta -bonds positioning near the chain terminus is highly ordered. Consequently, the central region of the bilayer's hydrocarbon core becomes less dynamic by the presence of omega 3 lipids. This is likely to promote locally a much more favorable environment for stronger lipid/protein lateral interactions. Such an environment may be critical for the stability and/or the optimal function of certain bilayer spanning proteins.

    FOOTNOTES

* This research was supported in part by U. S. Public Health Service Grant GM-17452 from NIGMS, National Institutes of Health, Department of Health and Human Services.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, Box 440, Health Sciences Center, University of Virginia Charlottesville, VA 22908. Tel.: 804-924-5010; Fax: 804-924-5069; E-mail:ch9t{at}virginia.edu.

    ABBREVIATIONS

The abbreviations used are: Delta -bonds, cis carbon-carbon double bonds; PC, phosphatidylcholine; PE, phosphatidylethanolamine; C(20):C(20)PE, saturated PE with 20 carbons in the sn-1 and 20 carbons in the sn-2 acyl chains; C(20):C(20:1Delta n)PE, monounsaturated PE with a saturated sn-1 C20-acyl chain and a monounsaturated sn-2 C20-acyl chain with a cis carbon-carbon double bond at the nth carbon atom from the carbonyl end (Delta n); C(20):C(20:2Delta n,n+3)PE, PE with a saturated sn-1 C20-acyl chain and a dienoic sn-2 C20-acyl chain in which two methylene-interrupted Delta -bonds are at the n and (n + 3) carbon atoms from the carbonyl end; C(20):C(20:3Delta n, n+3, n+6)PE, C(20):C(20:4Delta n, n+3,n+6,n+9)PE, and C(20):C(20:5Delta n, n+3,n+6,n+9,n+12)PE, 3, 4, and 5, respectively, methylene-interrupted Delta -bonds in the sn-2 C20-acyl chains; DSC, differential scanning calorimetry; MM, molecular mechanics; Tm, phase transition temperature; Delta T1/2, transition peak width at half-maximal height; ATS, all-trans segment.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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