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

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 novosynthesized 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 thesn-2 acyl chain of C(20):C(20)PE can affect the gel-to-liquid crystalline phase transition temperature,T m , of the lipid bilayer in some characteristic ways; moreover, the effect depends critically on the position ofcis double bonds in the sn-2 acyl chain. Specifically, we have constructed a novel T m diagram for the 15 species of unsaturated PE, from which the effects of the number and the position of cis double bonds onT m can be examined simultaneously in a simple, direct, and unifying manner. Interestingly, the characteristicT m 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.

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 C 20 fatty acids comprised of single or multiple methylene-interrupted cis double bonds. Subsequently, 15 molecular species of phosphatidylethanolamine (PE) with sn-1 C 20 -saturated and sn-2 C 20unsaturated acyl chains were semi-synthesized by acylation of C 20 -lysophosphatidylcholine with unsaturated C 20 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 H 2 O, were investigated by high resolution differential scanning calorimetry. In addition, the energy-minimized structures of these sn-1 C 20 -saturated/sn-2 C 20 -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, T m , 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 T m diagram for the 15 species of unsaturated PE, from which the effects of the number and the position of cis double bonds on T m can be examined simultaneously in a simple, direct, and unifying manner. Interestingly, the characteristic T m 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.
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 (⌬-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 carboncarbon double bonds are invariably separated by a three-carbon unit comprised of a methylene group (-CH 2 -) 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 (T m ) of the lipid bilayer. Specifically, Keough and associates (1)(2)(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(20:1⌬ 11 )PC, which has a considerably lower T m 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(20:2⌬ 11,14 )PC with a further reduction in T m . Interestingly, the introduction of a third cis double bond at C (17) results in a small increase in T m for C (20):C(20:3⌬ 11,14,17 )PC. This down and up trend in T m has been confirmed calorimetrically by other groups (4,5). One can immediately raise a relevant question as to whether this down and up T m profile is a special or a general characteristic for acyl chain unsaturation. Phrased differently, what kind of T m profile will be observed if the first ⌬-bond is introduced at C(5) or C (17) in the sn-2 acyl chain followed by successive incorporations of ⌬-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 T m profile in terms of molecular structures of unsaturated phospholipids? In order to find answers to these fundamental questions, phospholipids with ⌬-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 Ͻ T m have to be estimated.
Recently, we have semi-synthesized a limited number of diacyl mixed-chain phosphatidylethanolamines (PE) with sn-1 C 20 -saturated and sn-2 C 20 -unsaturated acyl chains (6 -9). Our calorimetric data showed that the T m profile exhibited by a series of mixed-chain PE containing 1-3 cis ⌬-bonds in the sn-2 C 20 -acyl chains at ⌬ 11 -, ⌬ 11,14 -, and ⌬ 11,14,17 -positions, respectively, is parallel to that displayed by the corresponding mixedchain PC observed earlier by Keough et al. (8). The influence of single and multiple ⌬-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 T m 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 C 20 -saturated and sn-2 C 20 -unsaturated acyl chains to a total of 15 different species. The phase transition behavior of these mixed-chain PE, in excess H 2 O, has also been investigated by high resolution DSC. Based on the calorimetric data, a novel T m diagram is generated for the first time. In this diagram, T m values of 9 series of mixed-chain PE each containing three or more lipids are systematically arranged. The shapes of T m profiles displayed by various lipids in all nine series can be seen simultaneously in plots derived from the T m 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 T m 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
Chemicals-With the exception of six species of unsaturated C 20 fatty acids that were synthesized in this laboratory as described in the next paragraph, all other C 20 -unsaturated fatty acids including arachidonic acid were obtained from Sigma. Lysophosphatidylcholine with a C 20 -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 C 20 Fatty Acids and Semi-synthesis of Mixed-chain PE-In the present investigation, six species of C 20 -unsaturated fatty acids were synthesized; they were cis-14-eicosenoic, cis-17eicosenoic, 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-14eicosenoic acid as an example, this method can be briefly described as follows: the starting material is 1-bromoundecan-11-ol. After the hy-droxyl 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 -hydroxyl alkyne. Upon further reacting with CH 2 (CO 2 Et) 2 in the presence of EtONa, the chain elongation step gives a product of appropriate total number of carbons, viz. the eicosa-14ynoic acid. Finally, the triple bond of this C 20 -alkynoic acid is cishydrogenated using Lindlar catalyst to form cis-14-eicosenoic acid. The syntheses of various dienoic C 20 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-cistetraenoic acid. The purity of the synthesized mono-and dienoic acids was estimated by high pressure liquid chromatography.
Mixed-chain sn-1 C 20 -saturated/sn-2 C 20 -unsaturated PE was semisynthesized 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 C 20 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 N 2 to avoid possible lipid oxidation. All lipids were purified by column chromatography on silica gel 60, with which a mixture of CHCl 3 , CH 3 OH, 5% NH 4 OH, 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 mol per sample was loaded on the thin layer plate and developed in CHCl 3 , CH 3 OH, 5% NH 4 OH (65:30:5). Prior to use, the lipid powder obtained from lyophilization of the lipid/benzene solution was kept at Ϫ20°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 NaN 3 . 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, T m , corresponds to the peak position with maximal peak height, and the transition enthalpy, ⌬H, can be determined from the area under the transition peak and the lipid concentration using the software provided by Microcal. In general, the T m value obtained at the transition with maximal peak height from the second DSC heating run was reproduced at Ϯ 0.1°C for each lipid sample. The ⌬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)(18)(19). It should be mentioned, however, that prior to stochastic search for the energyminimized 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 Ϫ ⌬s ϩ s ϩ ⌬s Ϫ (or s ϩ ⌬s Ϫ s Ϫ ⌬s ϩ ) as the added sequence (18), where s Ϯ refers to skew (Ϯ) conformations with torsion angles of about Ϯ 110 o and ⌬ denotes cis double bond with torsion angle of about 0 o . These initial coordinates of the crude structural model were used as a set of initial input data in MM3 computations.

RESULTS
The Phase Transition Behavior of Lipid Bilayers Composed of PE with sn-1 Saturated C 20 and sn-2 Unsaturated 3(or 6)C 20 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 3 derivatives. These unsaturated 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 3position) or 17 carbons from the carbonyl end (the ⌬ 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, T m , 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 3 (or ⌬ 17 )-position, the resulting C (20):C(20:1⌬ 17 )PE also displays a single, sharp endothermic transition; however, its T m 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 T m (Fig. 1). Interestingly, in the plot of T m versus the number of double bonds as illustrated in the inset of  Table I. In Fig. 2, the second DSC heating curves for aqueous dispersions prepared from four unsaturated 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 T m value. Moreover, a nonlinearly decreased T m curve in the plot of T m 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 allcis- 5,8,11,14-eicosatetraenoyl) chain has the smallest T m value of 6.6°C. It should be mentioned that the phase transition behavior of C (20):C(20)PE and some of its 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(20:1⌬ 14 )PE was missing. In contrast, Fig. 2 comprises all DSC thermograms for PEs with sn-1 C 20 -saturated/ sn-2 C 20 -6-unsaturated acyl chains. In Table I  base-exchange reaction in the presence of excessive ethanolamine. Furthermore, the monoenoic 6 fatty acid (cis-14-eicosenoic acid) was also synthesized in this laboratory. The single and symmetric endothermic transition with T m ϭ 47. The T m Diagram for PE with sn-1 C 20 -saturated/sn-2 C 20unsaturated Acyl Chains-In recent years, the phase transition behavior of six lipid species of C (20):C(20)PE derivatives with 9, 12, and 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 T m values from these six PEs and those obtained with 3 and 6 PEs shown in Figs. 1 and 2 are codified, a general T m diagram for sn-1 C 20 -saturated/sn-2 C 20 -unsaturated PE can be generated for the first time (Fig. 3). Specifically, the T m diagram shown in Fig. 3 has the shape of a right-angled triangle, comprising 15 species of sn-1 C 20 -saturated/sn-2 C 20 -unsaturated PE. These lipids are arranged into 5 levels depending on the position of the -carbon, where the -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: 15PE, 12PE, 9PE, 6PE, and 3PE. Furthermore, the unique T m value of any given sn-1 C 20 -saturated/ sn-2 C 20 -unsaturated PE is shown under the abbreviated name of the given lipid species in Fig. 3. Vertically, each column in the T m diagram also represents a series of unsaturated PEs, which share a common ⌬ n -bond. Hence, each series is designated as the ⌬ n PE series, where the superscript n denotes the position of the common cis carbon-carbon double bond (⌬-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 T m 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.
For lipid species aligned horizontally along each row in the T m 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 T m diagram shows another common feature exhibited by each series of the (3-12)PE as follows. The T m 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 T m diagram also show a growing number of cis double bonds in the sn-2 acyl chain of the lipid (Fig. 3). However, the methylene- The T m 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:⌬ 11 )PE, has the lowest T m value of 43.3°C; moreover, the T m 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 T m profile. Similar T m profiles can also be recognized for dienoic and trienoic series of PE as seen diagonally in the T m 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 chainmelting behavior. It should be mentioned that various V-shaped T m profiles for the monoenoic, dienoic, and trienoic series of PE shown in the plot of T m versus the position of cis double bonds have been documented previously, albeit separately, in the literature (6,8,9). Uniquely, the T m diagram illustrated in Fig. 3 shows simultaneously all these T m profiles in a simple and unifying manner. 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 C 20 -saturated/sn-2 C 20 -unsaturated acyl chains.
In Fig. 5, the energy-minimized structures of six lipid species in the 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 CH 2 -CH 3 bond is usually disordered at T Ͻ T m , particularly for lipids packed in the gel state bilayer. In the case of monounsaturated C(20):C(20:1⌬ 17 )PE, the sequence of the ⌬ 17 -containing kink in the sn-2 acyl chain is s Ϫ ⌬s Ϫ , where s Ϫ and ⌬ are skew(Ϫ) and cis double bonds, respectively. By MM calculations, a set of optimal torsion angles for this s Ϫ ⌬s Ϫ sequence is determined to be (Ϫ109°, Ϫ1.1°, and Ϫ120°) as indicated in Fig. 5. Consequently, the sn-2 acyl chain of C(20): C(20:1⌬ 17 )PE has a crankshaft-like topology in which a long and a short chain segments separated by the ⌬ 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(20:1⌬ 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 CϪC single bond preceding the ⌬ 17bond has a skew(Ϫ) conformation with an optimal torsion angle of Ϫ109°. For polyunsaturated 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 Ϯ and ⌬ 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 ⌬ 17 -bond is identical in length for all unsaturated 3PEs; in contrast, the length of ATS preceding the kink sequence in the sn-2 acyl chain decreases progressively with increasing numbers of ⌬-bonds.
The energy-minimized structures of the five lipids listed in the right column in the T m diagram (Fig. 3) have also been determined by MM calculations using the MM3 program (data not shown). Like lipids in the 3PE series shown in Fig. 5, lipids in this ⌬ 5 PE 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 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 ⌬ 5 -bond. The polyunsaturated lipids have their methylene-interrupted double bonds added on the methyl side of the ⌬ 5 -bond. As a result, the short chain segment of the kinked sn-2 acyl chain is invariable in length, 2 CϪC 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  In Fig. 7, the energy-minimized structures of lipids in the ⌬ 8 PE 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(20:2⌬ 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: 3⌬ 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(20:3⌬ 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 ⌬-bond is progressively introduced into the sn-2 acyl chain of the lipid. DISCUSSION 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 3 derivatives, it is appropriate to first comment on the simulated structures of these 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 Ͻ T m . 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 3PEs share a common chain termi-nal segment of C (16)ϪC (17)ϭC (18)ϪC (19)ϪC (20), in which the CϪC single bonds are all rotationally highly dynamic at T Ͻ T m . Hence, this disordered methyl-terminal segment does not undergo the thermally induced trans 3 gauche isomerizations at T m . 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 ⌬-bonds in this series of 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 T m 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 (⌬) is introduced into a long hydrocarbon chain, the six atoms in the immediate vicinity of the ⌬-bond, CϪCHϭCHϪC, are coplanar. Although the rotational flexibility of the ⌬-bond in the six-atom unit is highly restricted at physiological temperatures, paradoxically the CϪC single bond preceding or succeeding the ⌬-bond is rotationally highly flexible, in terms of torsion-angle fluctuations, even at very low temperature of Ϫ10°C (17). Hence, when a ⌬-bond is introduced into the sn-2 acyl chain of C(20): C(20)PE near either the carbonyl end at the ⌬ 5 -position or the methyl end at the 3-position, the short chain segment of the kinked sn-2 acyl chain in the gel-state bilayer at T Ͻ T m 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 T m involves principally the trans 3 gauche rotational isomerizations of methylene groups about CϪC 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 Ͻ T m , it thus makes no contributions to the chain disordering process at T m . 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 3 gauche isomerizations. When we compare the thermodynamic parameters (T m , ⌬H, and ⌬S) associated with the chain-melting phase transition for unsaturated lipids in the 3PE series at T Ͻ T m , 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 C 20acyl chain exists in all lipids in the 3PE series. Remember that the C-C double bond is rotationally highly restricted. We, therefore, also take the rigidity of multiple ⌬-bonds into consideration. Specifically, we suggest that the C-C double bond exerts its effect on the chain-melting phase transition when the ⌬-bond in the sn-2 C 20 -acyl chain reaches the number of three.
The changes in the chain length of ATS and the number of ⌬-bonds in the sn-2 acyl chain of the lipid can explain qualitatively the ⌬H trend observed with lipids in the 3PE series as shown in Table I. Here, the ⌬H values are seen to decrease initially with increasing number of ⌬-bonds. In particular, the ⌬H value is at a minimum for C (20):C(20:2⌬ 14,17 )PE with a sn-2 dienoyl chain; thereafter, ⌬H values are virtually independent of the number of ⌬-bonds. The transition enthalpy associated with the chain-melting transition of the bilayer is ⌬H ϭ H lc Ϫ H gel , 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 liquidcrystalline bilayers. We can thus assume that H lc is virtually identical for lipids with 0 -5 ⌬-bonds. As a result, the ⌬H trend exhibited by lipids in the 3PE series can, to a first approximation, be related to the H gel 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 ⌬ 17 -and ⌬ 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 ⌬H can thus be interpreted as follows: increasing up to two ⌬-bonds there is a steady increase in H gel as a result of progressive weakening of the overall chain-chain contact interactions. Beyond two ⌬-bonds, the rigid multiple methylene-interrupted ⌬-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 H gel . This enhanced contact interaction evidently is enough to compensate the opposing effect of ATS resulting from incorporation of additional ⌬-bonds. Consequently, for lipids with 3-5 ⌬-bonds in the 3PE series, their H gel and hence ⌬H values are nearly identical.
Similarly, the transition entropy for the chain-melting phase transition can be expressed as ⌬S ϭ S lc Ϫ S gel . For lipids in the 3PE series, the value of S lc can be assumed to decrease linearly with increasing number of ⌬-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 ⌬-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 ⌬-bonds on the S gel value cannot be linear. Specifically, as the ⌬-bond increases from 0 to 2, the rotational freedom of the lipid molecule as a whole or the S gel value increases markedly due to the high flexibility of C-C single bonds adjacent to the ⌬-bonds. Above 2, the S gel decreases with increasing ⌬-bonds as a result of the increased rigidity of multiple ⌬-bonds. In particular, the maximal S gel occurs at ⌬-bonds of 2, where the C-C single bonds adjacent to ⌬-bonds are highly flexible, whereas the overall rigidity of the two ⌬-bonds is not sufficient to cause a substantial decrease in S gel . Based on the proposed linear S lc curve and the nonlinear S gel curve in the plot of S versus the number of ⌬-bonds, a nonlinear ⌬S curve with a minimum of transition entropy at ⌬-bonds of 2 can be expected for lipids in the 3PE series. This expected ⌬S trend is indeed qualitatively similar to the calculated ⌬S values obtained with lipids in the 3PE series as shown in Table I.
For lipids in the 3PE series, the changes in T m as a function of alterations in the number of ⌬-bonds can now be considered. First, the following identity holds: T m ϭ ⌬H/⌬S. Second, despite the scattering of the data the ⌬H and ⌬S both change in the same direction as the number of ⌬-bonds varies. Specifically, the ⌬H and ⌬S both decrease markedly with increasing number of ⌬-bonds up to 2; thereafter, they increase slightly and then remain nearly unchanged (Table I). Based on these relationships and the T m profile observed in Fig. 1, we can conclude that the origin of T m is largely enthalpic, not entropic. Hence, for each lipid in the 3PE series the main contribution to T m is the overall lateral chain-chain attractive van der Walls interaction in the gel-state bilayer. However, in view of all lipids in the 3PE series, particularly those with 3-5 ⌬-bonds, the relative T m values must also be modulated differentially by entropic variations. As a result, the shape of the T m profile varies somewhat from that of the ⌬H profile. As discussed earlier, the chain-chain van der Walls attractive interaction depends largely on the length of ATS and the number of ⌬-bonds in the sn-2 acyl chain. In addition, the small contribution of the entropic effect also relates to the ATS and ⌬-bonds. Unfortunately, quantification of the relative contributions of ATS and ⌬-bonds to T m is not possible. Nevertheless, the relative magnitudes of T m for lipids in the 3PE series can be correlated qualitatively with the variations in the structural parameters of ATS and ⌬-bonds. We, therefore, propose that the nonlinear T m 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 T m lowering effect caused by the progressive shortening of ATS, and 2) the T m elevating effect exerted by the increasing rigidity of 3-5 ⌬-bonds. It is important to point out that the shortening of a three-carbon interval in the ATS has a more pronounced T m lowering effect than the opposing effect of the added ⌬-bonds for lipids in this series of 3PE. Consequently, the T m 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 ⌬-bonds on T m discussed above are also operative in bilayer membranes composed of other series of PE with 3 or more ⌬-bonds. The 6 and ⌬ 5 series of C (20): C(20)PE derivatives illustrated in the T m diagram (Fig. 3) can serve as examples. In each of the two PE series, the monounsaturated lipid has its ⌬-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 ⌬-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 T m . However, the magnitude of the T m reduction must be damped down somewhat due to an increasing number of ⌬-bonds beyond three. Specifically, the multiple ⌬-bonds in the sn-2 acyl chains of lipids in these two series of PE tend to promote higher T m , but this T m elevating effect is less than the T m lowering effect exerted by the shortening of the ATS in the sn-2 acyl chain. On balance, the T m values in the plot of T m versus the number of ⌬-bonds are expected to fall on nonlinearly decreasing curves for lipids from the 6-, or ⌬ 5 -PE series. This expectation is indeed borne out by experimental data (Fig. 2, inset, and Fig. 3).
The monounsaturated C (20):C(20:1⌬ 11 )PE is a common lipid species shared by the 9PE and the ⌬ 11 PE series as shown in the T m diagram (Fig. 3). The topological feature of C (20):C(20: 1⌬ 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 Ϫ ⌬s Ϫ g Ϫ . Here, the upper segment designates the chain segment closer to the bilayer/H 2 O 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: 1⌬ 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 ⌬-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 9PE and the ⌬ 11 PE series are obtained if the incorporation of ⌬-bonds takes place in the upper and lower segments of cis-11-eicosenoyl chain, respectively. Interestingly, the T m profiles exhibited by these two series of unsaturated PE are distinctly different (Fig. 4, A and B), indicating that the position of the incorporated ⌬-bond can markedly affect the chain-melting behavior. The shape of each of the two T m profiles will be interpreted later in terms of the length and location of ATS and the rigidity of the multiple ⌬-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 3 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 T m . 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 -CH 2units, then this short segment can most likely undergo the thermally induced trans 3 gauche isomerizations. It thus contributes to the overall chain-melting process. With these basic concepts in mind, we can rationalize the characteristic T m profile exhibited by lipids in the 9PE series.
The three lipids in the  5,8,11 )PE is observed calorimetrically to be smaller than that of C (20):C(20:2⌬ 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 3 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(20:2⌬ 8,11 )PE 3 C(20):C(20:3⌬ 5,8,11 )PE conversion, its contribution to the overall chain-melting process is totally abolished, resulting in a lower T m . However, all-cis-5,8,11-eicosatrienoyl chain has three ⌬-bonds, which can elevate the T m somewhat by retarding sterically the trans 3 gauche isomerizations of neighboring chains in bilayers. As a result, the ⌬T m between C(20):C(20: Thus far, we have discussed four T m profiles exhibited by lipids from 3-, 6-, 9-, and ⌬ 5 PE series. All four T m profiles show a similar pattern; the T m decreases nonlinearly as the number of ⌬-bonds in the sn-2 acyl chain of the lipid increases stepwise. Two other series of PE shown vertically in Fig. 3 are ⌬ 8 -and ⌬ 11 PE series; interestingly, the T m profile displayed by lipids from each of the ⌬ 8 -and ⌬ 11 PE series is characterized by a down and up pattern as illustrated in Fig. 4B. Each of the down and up T m profiles will be analyzed in the subsequent section; in particular, the structural features of those lipid species that give rise to higher T m upon further unsaturation will be delineated. Let us begin with the ⌬ 11 PE series. The energy-minimum structures of the three lipids in the ⌬ 11 PE 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 T m observed with lipids in the ⌬ 11 PE series; other structural features have to be considered. For C (20):C(20:1⌬ 11 )PE, the lower segment of the sn-2 acyl chain has 7 consecutive methylene units (Fig. 6I). Some of these -CH 2 -units, particularly those located far away from the chain methyl end, can make contributions to the chain-melting process of trans 3 gauche isomerizations, resulting in a higher T m . The other two lipids in the same series are C (20):C(20: 2⌬ 11,14 )PE and C (20):C(20:3⌬ 11,14,17 )PE, in which the short lower segments are relatively disordered at T Ͻ T m . Moreover, their lengths are smaller than the corresponding short segment in C (20):C(20:1⌬ 11 )PE by 3 and 6 C-C methylene units, respectively, as shown in Fig. 6. Among the three lipids in the ⌬ 11 PE series, C(20):C(20:1⌬ 11 )PE must, therefore, exhibit the highest T m . Next, we continue the comparison between C(20):C(20: 2⌬ 11,14 )PE and C (20):C(20:3⌬ 11,14,17 )PE. Structurally, the fundamental difference between them lies in the number of ⌬-bonds. Earlier, we have postulated that the presence of three to five cis ⌬-bonds in the sn-2 C 20 -acyl chain of PE can cause the bilayer to exhibit a higher T m . As before, this T m 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(20:2⌬ 11,14 )PE 3 C(20):C(20:3⌬ 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 3 gauche isomerizations underlying the main phase transition at T m . In going from C(20):C(20:2⌬ 11,14 )PE to C(20):C(20:3⌬ 11,14,17 )PE, a ⌬-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 -CH 2 -units upon unsaturation at C (17) will not affect appreciably the T m . As a result, the opposing effect of the rigid triple ⌬-bonds of C (20):C(20:3⌬ 11,14,17 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 ⌬-bond into all-cis-8,11,14eicosatrienoyl 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 Three series of mixed-chain PEs with fixed numbers of ⌬-bonds can be seen along the diagonal lines in the T m 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 T m values of lipids with a fixed number of double bonds give rise to a roughly V-shaped T m profile (Fig. 4,  A and B). Molecular interpretations of such a characteristic T m profile have been given in detail elsewhere from this laboratory (9). Hence, we shall not discuss the V-shaped T m 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 ⌬-bonds, a continuously decreasing T m profile is generally observed in the plot of T m versus the number of ⌬-bonds as exemplified by data  8,11,14,17 )PE conversions are coupled with increased T m , and the T m profiles observed with lipids in the ⌬ 8 -and ⌬ 11 PE series are thus characterized by a down and up trend. The mixed-chain PE that can, upon unsaturation, convert into a higher T m species has the following structural characteristics: 1) the sn-2 acyl chain contains at least two methylene-interrupted cis ⌬-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 ⌬-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 3 lipids such as C (20):C(20:3⌬ 11,14,17 )PE and C (20):C(20:4⌬ 8,11,14,17 )PE exhibit higher T m values than their 6 precursors as shown calorimetrically in the present and previous studies (8,23). The significance of the down and up T m profile is that it means the polyunsaturated 3 lipid with its multiple ⌬-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 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.