©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phase Transition Behavior and Molecular Structures of Monounsaturated Phosphatidylcholines
CALORIMETRIC STUDIES AND MOLECULAR MECHANICS SIMULATIONS (*)

(Received for publication, October 6, 1994)

Zhao-qing Wang Hai-nan Lin Shusen Li Ching-hsien Huang (§)

From the Department of Biochemistry, Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

High resolution differential scanning calorimetric studies were performed to investigate the thermotropic phase behavior of 26 molecular species of sn-1 saturated/sn-2 monounsaturated phosphatidylcholines. In parallel with calorimetric studies, the energy-minimized structures and steric energies of the diglyceride moieties of these monoenoic lipids were determined using a molecular mechanics approach. The combined calorimetric and computational studies led to the following results and conclusions. (i) When a single cis-carbon-carbon double bond (Delta) is incorporated into a saturated diacylphosphatidylcholine molecule at any position within the central segment of the long sn-2 acyl chain, the resulting monoenoic lipid molecules will, in excess water, exhibit reduced phase transition temperature (T) and transition enthalpy (DeltaH) as they undergo the gel to liquid-crystalline phase transition. The T and DeltaH-lowering effects of the Delta bond can be attributed to a decrease in the chain length of the sn-2 acyl chain, a change in the chain length difference between the sn-1 and sn-2 acyl chains, and a local perturbation of the chain-chain van der Waals interaction in the vicinity of the Delta bond. (ii) For a series of positional isomers of 1-stearoyl-2-cis-octadecenoylphosphatidylcholine, C(18):C(18:1Delta)PC, with a Delta bond at different positions along the sn-2 acyl chain, the T value depends critically on the position of the Delta bond. Specifically, the T value is minimal as the Delta bond is located at the geometric center of the linear segment of the sn-2 acyl chain, and the Tvalue is progressively increased as the Delta bond migrates toward either end of the sn-2 acyl chain. (iii) The various monoenoic phosphatidylcholines under study can be divided into two groups. The T values of most lipids in each group can be correlated in an identical manner with their structural parameters, yielding a commonT-structure relationship.


INTRODUCTION

Most biological membranes contain a substantial amount of glycerophospholipids loosely called phospholipids, and a remarkable feature of these membrane phospholipids is their bewildering diversity. This diversity originates largely from the various possible combinations of different fatty acyl chains present in the hydrophobic moiety of the amphipathic phospholipid molecule. Specifically, the structures of the two fatty acids hydrolyzed enzymatically from animal phospholipids display the following general features. (i) With the possible exceptions of mammalian lung and nerve endings, which contain large amounts of dipalmitoylphosphatidylcholine(1, 2) , identical fatty acids in both acyl positions occur rarely in a naturally occurring phospholipid molecule. Instead, the two fatty acids have a different even number of carbon atoms ranging from 14 to 22(3, 4) . (ii) The fatty acids in natural phospholipids are nonrandomly distributed. The one that is esterified to the hydroxyl group at glycerol backbone carbon 1, the sn-1 acyl chain, is often a saturated fatty acid, whereas the other, the sn-2 acyl chain, is predominantly an unsaturated one(3) . (iii) The unsaturated fatty acids derived from the sn-2 acyl chains of major membrane phospholipids in animal tissues have the structural formula CH(3)-(CH(2)) - [CH=CH-CH(2)] - (CH(2)) - COOH, where the subscripts alpha = 1, 4, 5, and 7, beta = 1-6, and = 2-7, and the cis-double bonds (beta = 1-6) are always separated by one methylene group(5) . Because of the large number of possible ways that permutations of chain length and unsaturation can occur, a given class of membrane phospholipids such as phosphatidylcholine isolated from a given cell type can be expected to give rise to a significant number of specific molecular species. Indeed, 57 molecular species of phosphatidylcholines have been detected in mammalian liver(6) . Probably because of the large number of molecular species of natural phosphatidylcholines, the physical properties of most membrane phosphatidylcholines with mixed sn-1 saturated/sn-2 unsaturated acyl chains have not yet been fully established. The thermotropic phase transitions of phospholipid bilayers and their relevance to biological membranes, for instance, have been investigated extensively for nearly 3 decades(7, 8) . Surprisingly, with the notable exception of 1-palmitoyl-2-oleoylphosphatidylcholine (9, 10, 11) and a few other species (10, 11) , only limited information is available regarding the phase transition behavior of most natural phosphatidylcholines. Clearly, a significantly rapid progress in this field is definitely needed.

The role played by amphipathic phosphatidylcholines in biological membranes has, until recently, been viewed primarily as that of a structural component of biological membranes in eukaryotic cells. This picture is now changed drastically, since the breakdown components of phosphatidylcholine molecule, or the intermediates of the ``phosphatidylcholine cycle,'' in the cell membrane have turned out to be involved actively in signal transduction in response to extracellular stimuli(12, 13) . In addition, there is increasing evidence suggesting that skeletal muscle phospholipids with polyunsaturated sn-2 acyl chains may be of great significance in modulating the insulin receptor activity(14, 15) . In view of these recent exciting findings, it is important to study systematically the various natural phosphatidylcholines with well defined composition and to obtain information regarding the specific contributions of the characteristic unsaturated acyl chains to the structure and properties of the lipid bilayer comprised of natural phosphatidylcholines.

As a first step to investigate systematically the physical properties of naturally occurring phosphatidylcholines with mixed sn-1 saturated/sn-2 unsaturated acyl chains, we have semisynthesized 26 molecular species of identical chain and mixed chain phosphatidylcholines, each containing a single cis-double bond in the sn-2 acyl chain. In this paper we report the phase transition characteristics of these molecular species as determined by high resolution differential scanning calorimetry (DSC). (^1)Moreover, molecular mechanics (MM) calculations are applied to refine the three-dimensional structures and to compute the steric energies of several series of Delta bond-containing phosphatidylcholine molecules which, in turn, are used to account for the thermodynamic data. Finally, the phase transition temperatures (T) associated with the gel to liquid-crystalline phase transitions of bilayers prepared from two groups of a total of 26 monoenoic phosphatidylcholines are analyzed in terms of their structural parameters by a multiple regression approach, leading to the derivation of two general equations. It is of the greatest interest and significance that there is a common T-structure relationship that holds for most different monoenoic phosphatidylcholines within each group of the natural lipids.


EXPERIMENTAL PROCEDURES

Chemicals

Monounsaturated fatty acids with various chain lengths, each containing a cis-double bond at different positions, were purchased from Sigma. Lysophosphatidylcholines with various saturated acyl chain lengths were provided by Avanti Polar Lipids, Inc. (Alabaster, AL). Silica Gel 60 (mesh 230-400) was obtained from EM Science (Gibbtown, NJ). All chemicals and organic solvents were of reagent and spectroscopic grades, respectively.

Semisynthesis of sn-1 Saturated/sn-2 Monounsaturated Phosphatidylcholines

Monounsaturated phosphatidylcholines were semisynthesized at room temperature by acylation of CdCl(2) adducts of lysophosphatidylcholine, in dry chloroform, with monounsaturated fatty acid anhydride that was prepared in situ from fatty acid and dicyclohexylcarbodiimide, in the presence of catalyst 4-pyrrolidinopyridine according to the modified procedure of Mena and Djerassi (16) as described previously(17) . The in situ reaction and the reacylation were carried out under an N(2) atmosphere to avoid the possible oxidation of the unsaturated fatty acid. The synthesized sn-1 saturated/sn-2 monounsaturated phosphatidylcholines were subsequently purified to about 99% purity by repeated column chromatography on Silica Gel 60(17) .

High Resolution DSC Measurements

Purified monoenoic phosphatidylcholines were lyophilized from benzene before weighing. The lyophilized sample was then dispersed in buffered (pH 7.4) aqueous solution containing 50 mM NaCl, 1 mM EDTA, and 5 mM phosphate buffer to give a final lipid concentration in the range of 3.0-6.0 mM. The exact lipid concentrations were determined by phosphorus analysis. Prior to DSC experiments, the lipid sample was kept in the cold room (0 °C) overnight under an N(2) atmosphere. DSC experiments were carried out with a high resolution Microcal calorimeter (model MC-2) equipped with DA-2 digital interface and data acquisition utility for automatic collection (Microcal, Northampton, MA). After loading the lipid sample into the sample cell of the calorimeter, it was preincubated in the calorimeter for a minimum of 60 min at the desired temperature (usually 15 °C below the estimated T(m)) followed by the DSC heating scan. The scan was terminated at about 15 °C above the estimated T(m). The sample was then allowed to equilibrate thermally at the elevated temperature for 60 min prior to the cooling scan. Each sample was scanned at least five times: three heating and two cooling runs. In all of these experiments, a nominal scan rate of 15 °C/h was used. The T(m) and the transition enthalpy (DeltaH) were determined, as described in(17) , from the second and third heating curves, and the average values were reported for each sample.

MM Calculations

The MM2 force field (Version 85), written by Allinger (18) and supplied by Quantum Chemistry Program Exchange (Department of Chemistry, Indiana University), was chosen as the software for the MM calculations. All MM computations were run on an IBM RS/6000 computer workstation. The structural data from the output of the MM2 calculations were transferred and then displayed in the form of various two-dimensional representations of the three-dimensional molecular structure, which was accomplished by the computer-assisted molecular graphics using the software package HyperChem (Autodesk Inc., Sausalito, CA) performed on a 486 platform.

Prior to MM calculations, a set of torsion angles for a crude representation of the molecule under study was first constructed. This starting set of atomic coordinates was entered, via the MM2 program, into the computer. An optimized structure corresponding to the minimum steric energy (E(s)) closest to the starting point was subsequently determined for the molecule by MM calculations using a set of potential energy functions (called force fields). These calculations involved a series of repeated automatic cycles of the second derivative Newton-Raphson method for energy minimizations. These cycles of self-adjusted computations came to a halt as the steric energy difference between two consecutive cycles was less than (0.00008 times molecular weight) kcal/mol. For the MM2 program, the steric energy has the following terms: E(s) = E + E(b) + E + E + E + E, where E = bond stretch energy, E(b) = bond angle bend energy, E = torsional energy, E = bond dipole interaction energy, E = van der Waals nonbonding interaction energy, E = coupling energy between bond stretching and bond bending.

It should be emphasized that the absolute value of E(s) is of no great significance, since it depends on the software program used. When the same MM2 program is adopted, however, the E(s) values for a homologous series of positional isomers are quite useful for making relative comparisons.

The initial crude structure of C(16):C(18:1Delta^9)PC was constructed based on the energy-minimized structure of C(14):C(14)PC (19) , and the torsion angles of oleate chain of cholesteryl oleate were determined by x-ray diffraction(20) , as described previously(21) . Briefly, the torsion angles of the sn-2 acyl chain of C(16):C(18:1Delta^9)PC starting from C(3) to the chain methyl terminus were taken from the crystal data of the cholesteryl oleate, and all other torsion angles of the lipid molecule were taken from the energy-minimized structure of C(14):C(14)PC except that the sn-1 acyl chain was extended by two methylene units in the trans form. This assembled structure was taken as the crude structure of C(16):C(18:1Delta^9)PC. It was subsequently subjected to MM calculations in which the internal coordinates were modified by Allinger's MM2 program until a minimum in the potential energy surface was reached. The resulting structure was taken as the optimized or energy-minimized structure of C(16):C(18:1Delta^9)PC. The torsion angles of the various bonds, most importantly those in the vicinity of the Delta bond, obtained with this optimized structure were used subsequently as the starting atomic coordinates to search for minimal energy structures of other monoenoic lipids with the Delta bond at different positions in the sn-2 acyl chain. For instance, the initial crude structure of C(18):C(18:1Delta)PC can be readily created by adding two methylene units in the trans form into the sn-1 acyl chain of C(16):C(18:1Delta^9)PC and by moving the Delta bond to the Delta position in the sn-2 acyl chain. The refined three-dimensional energy-minimized structure of C(18):C(18:1Delta)PC can then be obtained by subjecting the crude structure to MM calculations using the MM2 program. Similarly, the same protocol can be used to obtain energy-minimized structures of other monoenoic phosphatidylcholines. However, when a Delta bond was introduced into the sn-2 acyl chain at an even carbon atom such as C(18):C(18:1Delta)PC, the initial values of the two torsion angles immediately preceding the Delta bond ((1) and (2)) and those (`(1) and `(2)) immediately succeeding the Delta bond in the sequence (2)(1)Delta`(1)`(2) were assigned to be negative, although the absolute magnitudes of these torsion angles were still taken from those of the energy-minimized structure of C(16):C(18:1Delta^9)PC. This change in sign was simply to reflect the fact that even and odd carbons in an all trans-acyl chains lie commonly on two opposite and parallel planes.

In this paper, attempts have been made to compare the structures of various phosphatidylcholines. Since all of these lipid molecules have the same head group, being different in their diglyceride moieties only, we feel that it is justified to simplify our MM calculations by considering the diglyceride moieties of these lipid molecules only. Consequently, various sn-1 saturated/sn-2 unsaturated phosphatidylcholines without the headgroups are used exclusively for our MM calculations. In this approach, the headgroup-headgroup interactions are assumed to be identical, and the headgroup-diglyceride interactions are assumed to be small; hence, they are ignored when a relative comparison among phosphatidylcholines is made. Furthermore, the water molecules are considered to be excluded from the hydrophobic interior; hence, Allinger's MM2 calculations of the diglyceride moiety of the lipid molecule were performed in vacuo to avoid solvent interactions.

For dimers and tetramers, the MM calculations were performed according to the same procedure as that used for the monomer, essentially as described in detail elsewhere(19) . Again, these aggregates are the aggregated diglyceride moieties of the lipid molecules.


RESULTS

Effect of the Position of cis-Double Bond on the Phase Transition Behavior of 1-Stearoyl-2-octadecenoyl-sn-glycerol-3-phosphocholine (C(18):C(18:1Delta^n)PC)

The DSC heating thermograms for aqueous dispersions of six isomers of C(18):C(18:1Delta^n)PC are presented in Fig. 1. These positional isomers with the same molecular weight have a common sn-1 acyl chain. In addition, each has only one cis-carbon-carbon double bond (Delta) in the sn-2 octadecenoyl chain. However, the position of the double bond from the carboxyl carbon of the acyl chain (Delta^n) is different, with n = 6, 7, 9, 11, 12, and 13. Each DSC heating thermogram shown in Fig. 1is characterized by a sharp, single, nearly symmetrical, endothermic transition that is reproducible upon subsequent reheatings. The observed transition is assigned as the gel to liquid-crystalline phase transition or the chain melting transition. The T(m) and DeltaH associated with the chain melting transition for various isomers of C(18):C(18:1Delta^n)PC, in excess water, are summarized in Table 1. The T(m) values obtained with the cooling exotherms (data not shown) of these lipid samples occur at temperatures that are either identical to or slightly lower (0.2-0.4 °C) than the T(m) values obtained with the heating endotherms.


Figure 1: Representative DSC heating thermograms for C(18):C(18:1Delta^6)PC, C(18):C(18:1Delta^7)PC, C(18):C(18:1Delta^9)PC, C(18):C(18:1Delta)PC, C(18):C(18:1Delta)PC, and C(18):C(18:1Delta)PC dispersions. The T, DeltaH, and DeltaT values associated with the gel to liquid-crystalline phase transition are given for each lipid sample. All thermograms were the second DSC heating scans obtained at a constant scan rate of 15 °C/h.





It is interesting to recognize from Fig. 1that as the cis-double bond migrates from the carboxyl end toward the chain methyl terminus, the phase transition peaks are changed accordingly both in terms of the peak width and the position of the maximal peak height (T(m)). Specifically, the transition peak width at half-height (DeltaT) is seen to increase from 0.3 to 1.1 °C as the position of the Delta bond (Delta^n) moves from C(6) to C(13); the T(m) value decreases steadily from 24.8 °C to a minimum value of 3.8 °C as the Delta bond moves along the chain from C(6) to C(11) and then increases progressively from 3.8 to 15.9 °C as the Delta bond moves further along the chain from C(11) to C(13). Most interestingly, the T(m) values in the T(m)versus Delta^n plot shown in Fig. 2can be fitted by the least squares procedure to a smooth parabolic curve with a correlation coefficient of 0.9951. In comparison, the DeltaH values are, within experimental errors, relatively insensitive to the position of the Delta bond (Table 1), but a general trend similar to that of the T(m) profile is evident.


Figure 2: Tversus Delta for C(18):C(18:1Delta)PC. The experimental values of T are indicated by open circles, and the solid line connecting the experimental data is the least squares line of a parabola best fit for the data. For comparison, the dotted line connecting the T values of C(18):C(18:1Delta)PE (PE) obtained earlier from this laboratory (23) is included.



The least squares fitting curve in the T(m)versus Delta^n plot yields Delta^n = 10 and T(m) = 3.8 °C as the abscissa and ordinate of vertex, respectively, for monoenoic C(18):C(18:1Delta^n)PC (Fig. 2). The calorimetric T(m) value for aqueous dispersion of saturated C(18):C(18)PC is 55.3 °C(22) . The calculated T(m) value for C(18):C(18:1Delta)PC based on the coordinates of vertex of the best fit parabola shown in Fig. 2is 3.8 °C, corresponding to a significant drop in absolute temperature of 15.7%. However, as the Delta bond moves toward either end of the sn-2 acyl chain, the T(m)-lowering effect is diminished considerably. The identification of an inverted bell-shaped relationship between the T(m) values of monoenoic phosphatidylcholines and the different positions of the cis-double bond along the sn-2 acyl chain should come as no surprise, since a similar but not identical relationship with the lowest T(m) value occurring at the Delta bond position of C(10) has also been observed for the lipid bilayers of a different series of chemically distinct C(18):C(18:1Delta^n)PE (23) . For comparison, the statistical least squares line best fit to the calorimetric data obtained with C(18):C(18:1Delta^n)PE is presented in Fig. 2as a dotted line.

Effect of Increasing sn-1 Acyl Chain Length on the Phase Transition Behavior of Mixed Chain Monounsaturated Phosphatidylcholines

We have semisynthesized five additional series of sn-1 saturated/sn-2 monounsaturated phosphatidylcholines, and the aqueous dispersions prepared from these five series of lipids have been subsequently studied calorimetrically. Within each of the five series, the sn-1 acyl chain length is increased stepwise by two methylene units, but the chemical composition of the sn-2 acyl chain is fixed. Fig. 3illustrates the second DSC heating scans for aqueous dispersions of four lipid samples, which represent one of the five series of monoenoic lipids under study. In this series of examples, the sn-1 acyl chains of the four lipids are derived from stearic, arachidic, behenic, and lignoceric acids, respectively, whereas the sn-2 acyl chains are derived commonly from gondoic acids or 11-eicosenoic acids (20:1Delta). Fig. 3shows clearly a distinct gel to liquid-crystalline phase transition exhibited by each sample. In this figure, the T(m) and DeltaH values associated with the endothermic transition are observed to increase progressively with increasing sn-1 acyl chain length. Among the four lipid species shown in Fig. 3, C(20):C(20:1Delta)PC is the only one that has been studied previously by DSC(24) . The T(m) and DeltaH values obtained from Fig. 3for C(20):C(20:1Delta)PC are 19.8 °C and 7.5 kcal/mol, respectively, which are comparable to those reported in the literature (24) .


Figure 3: Representative DSC heating thermograms for a series of C(X):C(20:1Delta)PC. As the sn-1 acyl chain length is increased successively by two methylene units, the T and DeltaH values are also seen to increase progressively. The values of DeltaH, T, and DeltaT are given for each sample.



Aqueous lipid dispersions prepared from four other series of sn-1 saturated/sn-2 monounsaturated phosphatidylcholines in which the sn-2 acyl chains are derived from petroselenic, oleic, erucic, and nervonic acids have also been studied calorimetrically. The thermodynamic data (T(m), DeltaH, DeltaS, and DeltaT) associated with the gel to liquid-crystalline phase transition exhibited by aqueous dispersions prepared individually from these lipids and lipids of the first series (Fig. 3) are listed in Table 1. In Fig. 4, the T(m) value is plotted against the total number of carbon atoms in the saturated sn-1 acyl chain for each of these monoenoic lipids. Clearly, the T(m) value is observed to increase with increasing total number of carbon atoms in the sn-1 acyl chain for all five series of monoenoic lipids. This general trend reflects that an increase in the sn-1 acyl chain length is accompanied by an increase in the stability of the lipid bilayer in the gel state.


Figure 4: T values versus the total number of carbon atoms in the sn-1 acyl chain for monoenoic phosphatidylcholines. In this figure, experimental data from five series of lipids each with a common monounsaturated sn-2 acyl chain are plotted. The solid lines connect all experimental T values within each lipid series.



Upon a closer inspection of Fig. 4, the T(m) curves for C(X):C(18:1Delta^9)PC, C(X):C(20:1Delta)PC, C(X):C(22:1Delta)PC, and C(X):C(24:1Delta)PC show that the larger the number of carbon atoms that can be found in the sn-2 acyl chain, the higher the T(m) curve for a given lipid series. However, the correlation between the T(m) curve and the total number of carbon atoms in the sn-2 acyl chain, based on the shape of the T(m) curve, is not perfect. Furthermore, the T(m) curve exhibited by aqueous dispersions prepared from the series of C(X):C(18:1Delta^6)PC, shown in Fig. 4, appears to be completely out of place among other T(m) curves. Recall the chain melting studies of a homologous series of lipids with different positions of the Delta bond (Fig. 2); the misplaced curve shown in Fig. 4is thus most likely the result of the influence of the position of the Delta bond. In the case of C(X):C(18:1Delta^6)PC, the Delta bond is positioned three C-C bond lengths away from C(10), the geometric center of the linear segment of sn-2 acyl chain. The Delta bond in the series of C(X):C(18:1Delta^9)PC, however, lies commonly at the geometric center of their sn-2 acyl chains. In fact, the other two series of lipids also have their Delta bonds located at or near the geometric center of the sn-2 acyl chains. In summary, experimental data shown in Fig. 4indicate that the T(m) value of the lipid bilayer comprised of sn-1 saturated/sn-2 monounsaturated phosphatidylcholines depends on the total number of carbon atoms in both acyl chains as well as the position of the Delta bond along the sn-2 acyl chain.

The DeltaH values exhibited by various sn-1 saturated/sn-2 monounsaturated phosphatidylcholines can be grouped according to the total number of carbon atoms in the two acyl chains of the lipid structure. The results are given in Table 2. They show that the DeltaH value does not, within the large experimental error, vary greatly for each member of the same group with a common molecular weight (or total number of carbon atoms). As a result, the weight average transition enthalpy (DeltaH) can be calculated from the experimental data for each group of the sn-1 saturated/sn-2 monounsaturated phosphatidylcholines as shown in Table 2. Comparing DeltaH values among different groups shows, however, that the DeltaH for each group of monoenoic lipids can be best fit by a least squares line (Fig. 5), yielding DeltaH (kcal/mol) = 0.25 [C(X)+C(Y)] - 2.37, with a correlation coefficient of 0.9952, where C(X) and C(Y) are the total number of carbon atoms in the sn-1 and sn-2 acyl chains of the monoenoic lipid, respectively. When C(X) = C(Y), this linear function can be subtracted from another linear function derived from the experimental DeltaH values obtained with lipid bilayers comprised of saturated identical chain phosphatidylcholines (shown as a dashed line in Fig. 5), leading to a new linear function (Fig. 5, inset) with the expression DeltaDeltaH (kcal/mol) = 0.23 [C(X) + C(X)] - 5.26 = 0.46 C(X) - 5.26. Here, the term DeltaDeltaH specifies the decrease in DeltaH as a cis-double bond is introduced into the identical chain phosphatidylcholine's sn-2 acyl chain. For instance, the experimental DeltaH value associated with the gel to liquid-crystalline phase transition of C(18):C(18)PC bilayers is 9.8 kcal/mol(25) ; the DeltaDeltaH value can be calculated as 0.46 (18) - 5.26 = 3.0 kcal/mol. The DeltaH value associated with the gel to liquid-crystalline phase transitions of C(18):C(18:1Delta^n)PC bilayers (n = 6, 7, . . 13) can thus be estimated as 9.8 - 3.0 = 6.8 kcal/mol. It should be emphasized that this equation, DeltaDeltaH = 0.46 C(X) - 5.26, can be used not only to estimate the weight average DeltaH value for the positional isomers of monoenoic phosphatidylcholines, it also indicates that DeltaDeltaH = 0 when the total number of carbon atoms in the sn-1 or the sn-2 acyl chain of the identical chain phosphatidylcholine approaches 11. This implies that short chain C(11):C(11)PC bilayers are already quite disordered at T < T(m), and as a consequence the introduction of a cis-double bond into the sn-2 acyl chain will not further disrupt and destabilize the gel state bilayer of C(11):C(11)PC.




Figure 5: DeltaHversus the total number of carbon atoms in both acyl chains of monoenoic lipids. The DeltaH value is the weight-averaged DeltaH value obtained with aqueous dispersions of monoenoic lipids, C(X):C(Y:1Delta)PC, with the same molecular weight as shown in Table 2. The dashed line connects the DeltaH values obtained with aqueous dispersions of saturated identical chain phosphatidylcholines, C(X):C(X)PC, reported by Lewis et al.(25) . The difference in DeltaH values between the dashed and solid lines (DeltaDeltaH) is plotted against the total number of carbon atoms in both acyl chains of phospholipids in the upper left inset.



MM Computations of Monoenoic Series of C(18):C(18:1Delta^n)PC with n = 6, 7, 9, 11, 12, and 13

In a recent study, the energy-minimized structures of C(16):C(18:1Delta^9)PC with various motifs of the crankshaft-like kink in the sn-2 acyl chain have been identified(21) . Specifically, four general groups of the crankshaft-like kinks have been derived from a common protocol for the sn-2 acyl chain of C(16):C(18:1Delta^9)PC, at T < T(m), by molecular mechanics calculations using Allinger's MM2 program. Within each group, four different kink-forming sequences are known; only minor variations in E(s) are detected among these four kink models within each group. The torsion angles of the various bonds in the immediate neighborhood of the Delta bond, or the kink-forming sequence, for a representative rotomer of C(16):C(18:1Delta^9)PC from these four groups are presented in Table 3. Most interestingly, the energy-minimized structure of the C(16):C(18:1Delta^9)PC rotomer with the lowest steric energy of 22.78 kcal/mol, shown in Table 3, is identical to the one derived independently using the torsion angles of the oleate chain of crystalline cholesteryl oleate followed by energy minimization using Allinger's MM2 program as described under ``Experimental Procedures.'' The atomic coordinates of this lowest-energy structure have thus been used as the basic set of structural data for constructing the initial three-dimensional structure of all other related members of monoenoic lipids as described under ``Experimental Procedures.'' Since the basic set of atomic coordinates is derived from crystal data, the computed energy-minimized structure presented in this paper should represent the lipid's simulated equilibrium structure at T < T(m).



Fig. 6illustrates the energy-minimized graphical structures of the diglyceride moieties of C(18):C(18:1Delta^n)PC with n = 6, 7, 9, 11, 12, and 13. For comparison, the energy-minimized graphical structure of the diglyceride moiety of C(18):C(18)PC with all-trans-acyl chains is also included in Fig. 6. In each of the graphical structures, the long axis joining all even carbons in the all-trans-sn-1 acyl chain is assigned as the x axis with the origin of the Cartesian coordinates fixed at C(4) and the positive direction of the x axis oriented toward the chain methyl terminus. The y axis is oriented from C(4) toward the midpoint of an imaginary line connecting C(3) and C(5) of the sn-1 acyl chain; the z axis is, then, perpendicular to the plane of the paper pointing toward the viewer. Several distinct features are revealed by the energy-minimized structures of monoenoic rotomers. (i) The sn-2 acyl chains of these monoenoic lipids are seen to have a crankshaft-like kink with average torsion angles for carbon-carbon bonds around the Delta bond ((2), (1), Delta, (1)`, (2)`) being (66.0° ± 2.3, 134.7° ± 6.4, 0.3° ± 0.4, 155.4° ± 3.0, 176.5° ± 0.3). The values of (2), (1), (1)`, and (2)` are positive for lipids with the Delta bond (negative value) positioned at the odd carbon, and negative for those with the Delta bond (positive value) at the even carbon (Table 4). (ii) In each structural model, the zigzag planes of the two segments of the sn-2 acyl chain separated by the kink are nearly parallel to the x-z plane, which is virtually perpendicular to the zigzag plane of the sn-1 acyl chain. However, the long chain axis of the all-trans-sn-1 acyl chain is nearly parallel to the chain axes of the two segments of the sn-2 acyl chain. The average spatial distance separating the two terminal methyl groups along the x axis direction (DeltaC) is 5.67 Å ± 0.3 or 4.46 ± 0.24 C-C bond lengths for this series of C(18):C(18:1Delta^n)PC. The corresponding DeltaC value for saturated C(18):C(18)PC can be calculated from the MM2 program to be 4.64 Å or 3.66 C-C bond lengths. Hence, the presence of a Delta-containing kink shortens the effective chain length of the sn-2 acyl chain by 1.03 Å or 0.81 C-C bond lengths. (iii) The number of trans-C-C bonds in the upper segment of the sn-2 acyl chain, C(18:1Delta^n), is (n - 5). In fact, the total number of C-C bonds in the upper segment is (n - 1). The first two C-C bonds are gauche, leading to a 90° bend in the sn-2 acyl chain. The two C-C bonds ((1) and (2)) immediately preceding the Delta bond are gauche and skew (Fig. 6). Consequently, the total number of trans-C-C bonds (180° ± 10) is (n - 1) - 2 - 2 = (n - 5). Similarly, it can be shown that the total number of trans-C-C bonds in the lower segment is (16 - n). These numbers, (C-C) and (C-C), are given in Fig. 6for each molecular species of the series of C(18):C(18:1Delta^n)PC. (iv) The E(s) for C(18):C(18:1Delta^7)PC, 23.75 kcal/mol, is the minimal value among all E(s) values calculated for this lipid series, and the E(s) of 24.55 kcal/mol for C(18):C(18:1Delta)PC is the maximal. The average value of E(s) is 24.19 ± 0.31 kcal/mol.


Figure 6: Energy-minimized structures of various phosphatidylcholine monomers. Panels A and A`) are two different representations of the same saturated distearoylphosphatidylcholine. Panels B-G are monoenoic lipids with the same molecular weight but with different position of the Delta bond. The local sequence around the Delta bond in the sn-2 acyl chain is expressed as gsDeltast and the torsion angles (in degrees) of the various bonds in the sequence are given under each energy-minimized structure after gsDeltast, where g is the gauche bond or conformation, t is the trans conformation, and s is the skew conformation of a C-C single bond. E denotes the steric energy and DeltaC the effective chain length difference between the two acyl chains. (C-C) and (C-C) denote the trans-carbon-carbon bond lengths in the upper and lower segments of the sn-2 acyl chain, respectively.





MM Computations of the Aggregates of Tetrameric C(18):C(18:1Delta^n)PC

Once the energy-minimized structures of the diglyceride moieties of monoenoic C(18):C(18:1Delta^n)PC were constructed as shown in Fig. 6, the Cartesian coordinates of these structures were then chosen as the new starting points to yield the energy-minimized tetramers with the front to back and the up and down motifs(19) . The aim was to combine these two types of energy-minimized tetramers in creating a simplified bilayer model in which the lipid molecules were packed in an orthorhombic lattice in the two-dimensional plane of the bilayer(19) .

Fig. 7illustrates the two types of the energy-minimized tetramers of the diglyceride moieties of C(18):C(18:1Delta^9)PC. The F-B packing motif is characterized by the superposition of two trans-bilayer dimers stacked in an eclipsed position, allowing van der Waals contact interactions to occur optimally between the two contiguous faces (Fig. 7A). The energy-minimized U-D tetramer is illustrated in Fig. 7B, in which two trans-bilayer dimers, lying on a common plane, are aligned side by side. In this packing motif, the dimer-dimer contact surface is limited to the lateral chain-chain interaction. The steric energies associated with the two types of tetrameric packings, after energy minimization by the MM2 program are also given in Fig. 7as E(t) and E(t), where the subscript t denotes the tetramer and the superscripts F-B and U-D denote the F-B and U-D packing motifs, respectively. It is apparent from Fig. 7that the general structural features of monoenoic lipids seen in Fig. 6are basically preserved in the two tetrameric motifs.


Figure 7: Energy-minimized structures of tetrameric C(18):C(18:1Delta^9)PC. Panel A, the tetramer with an F-B motif. Panel B, the tetramer with a U-D motif. The stabilization energy of the tetramer contributed by the monomer is expressed as DeltaE(t). These two energy terms are related to each other as follows: DeltaE(t) = (E(t) - 4E(m))/4, where E(m) is the steric energy of the monomer, corresponding to the E term seen in Fig. 6. Both E(t) and E(m) values are obtained by Allinger's MM2 program.



To get into more details, the stabilization energy of the tetramer contributed by the monomer (DeltaE(t)) has also been determined by MM calculations using the MM2 program. In general, the intermolecular interactions among four monomers within a tetramer are intimately associated with the stability of the tetramer. If the geometry of the monomer is such that extensive attractive interactions can occur among them, then this tetramer can be expected to be stable at equilibrium. The affinity of the four monomers for each other in the aggregated tetramer is a measure of the stabilization energy of the tetramer contributed by its constituent monomer, which can be calculated from the relationship DeltaE(t) = (E(t) - 4E(m))/4. The magnitudes of DeltaE(t) and DeltaE(t), given in Fig. 7, for the F-B and U-D motifs, are -19.32 and -8.75 kcal/mol, respectively. Clearly, the tetrameric C(18):C(18:1Delta^9)PC with an F-B packing motif is a more stable structure. If two tetramers, one with an F-B motif and the other with a U-D motif, are closely packed to mimic an orthorhombic two-dimensional lattice, then the overall averaged stabilization energy, DeltaE(t), is (DeltaE(t) + DeltaE(t)) = -14.04 kcal/mol.

The stabilization energy of the tetramer contributed by the monomer (DeltaE(t) + DeltaE(t)) and the overall averaged stabilization energy (DeltaE(t)) for C(18):C(18:1Delta^n)PC with n = 6, 7, 9, 11, 12, and 13 are listed in Table 5. Computational results indicate that the calculated -DeltaE(t) values for the simplified bilayer models of C(18):C(18:1Delta^n)PC are in the range of 14.03-14.41 kcal/mol with a mean of 14.18 ± 0.22; the largest difference between the mean and the calculated values amounts to only a few tenths of a kcal/mol. It is important to realize that various approximations have been made in our MM calculations in the determination of -DeltaE(t) values. Nevertheless, with the exception of C(18):C(18:1Delta)PC, the -DeltaE(t) values for all other C(18):C(18:1Delta^n)PC shown in Table 5appear to exhibit a V-shaped profile in the -DeltaE(t)versus Delta^n plot, with the minimum at Delta (Fig. 8C). Recall from Table 2that the experimental DeltaH values associated with the gel to liquid-crystalline phase transitions of C(18):C(18:1Delta^n)PC bilayers are in the range of 6.0 ± 0.7 to 7.1 ± 0.5 kcal/mol with a DeltaH value of 6.6 ± 0.7 kcal/mol and that the minimal DeltaH value occurs at Delta. The calculated -DeltaE(t) values resulting from MM computations thus harmonize with the experimental DeltaH values obtained calorimetrically (Fig. 8, B and C), indicating that the energy contents of C(18):C(18:1Delta^n)PC bilayers in the gel state are not greatly different and that the stability of the C(18):C(18:1Delta)PC bilayer appears to be the weakest.




Figure 8: Plots of various calorimetric and computational data versus the position of the Delta bond in C(18):C(18:1Delta)PC. The various notations used in the y axis of different plots are explained in the legends of Fig. 1and Fig. 6and in the legend of Table 4. Data in plots A and B are derived from DSC experiments; plots C and D are from MM calculations.




DISCUSSION

The first important overall result of our DSC studies is the quantitative demonstration that a single Delta bond has the profound effect of altering the gel to liquid-crystalline phase transition behavior of fully hydrated phosphatidylcholines. In particular, when a single Delta bond is incorporated into a long sn-2 acyl chain containing 18-24 carbons and is positioned at the carbon atom numbering from C(6) to C(15), the T(m) and DeltaH values associated with the phase transition are shown calorimetrically to decrease significantly. For example, the T(m) and DeltaH values for the C(18):C(18)PC bilayer are, respectively, 55.3 °C and 9.8 kcal/mol(25) . In contrast, the corresponding values for the C(18):C(18:1Delta^9)PC bilayer are 5.6 °C and 6.5 kcal/mol (Table 1). A reduction in the transition entropy (DeltaDeltaS) of 6.6 cal/molbulletK can thus be calculated. These fairly pronounced T(m)- and DeltaH-lowering effects of the Delta bond imply a decrease in the overall stability of the monoenoic gel state bilayer.

In parallel with calorimetric studies, our MM calculations provide structural evidence indicating that the introduction of a Delta bond into the sn-2 acyl chain allows the chain to kink in the shape of a crankshaft at T < T(m) ( Fig. 6and Fig. 7). This kink shortens the effective chain length of the sn-2 acyl chain by about 1.03 Å, which will undoubtedly diminish somewhat the stability of the gel state bilayer. Furthermore, a shortening of the sn-2 acyl chain may lead to an increase in the effective chain length difference between the sn-1 and sn-2 acyl chains (DeltaC), which has long been known to perturb the stability of the gel state bilayer(17) . In addition, the nearly uniform chain-chain separation distance is distorted by the kink; hence, favorable van der Waals interactions between acyl chains must be reduced to some extent. Our MM study thus indicates that a shortening of the sn-2 acyl chain, a change in the DeltaC value, and a perturbation in the overall chain-chain separation distance as induced by the Delta bond are three important factors that must be involved in the reduction in the overall average stabilization energy of the lipid aggregates (DeltaE(t)) and also in the depression of the DeltaH value associated with the lipid phase transition. In conclusion, it may be worthwhile to emphasize that when a single Delta bond is introduced locally into the sn-2 acyl chain, there is a decrease in the overall chain length in the sn-2 acyl chain within the lipid molecule. Associated with this chain length shortening, there are rotational isomerizations about the C-C bonds in the vicinity of the Delta bond which affect both the intra- and intermolecular van der Waals interactions in the gel state bilayer, thus producing a decrease in the stability of the entire lipid molecule in the gel state bilayer at T < T(m).

The second important set of results from our DSC studies indicates that the T(m) value exhibited by bilayers prepared from C(18):C(18:1Delta^n)PC depends critically on the position of the Delta bond in the sn-2 acyl chain (Delta^n). Notably, a V-shaped curve in the T(m)versus Delta^n plot with the minimal T(m) value occurring at Delta^n = 10 is observed (Fig. 2). This V-shaped profile is in excellent agreement with the recent calorimetric data obtained with aqueous lipid dispersions prepared from a series of sn-1 saturated/sn-2 monounsaturated C(18):C(18:1Delta^n)PE(23) . A similar V-shaped profile has also been reported earlier by Barton and Gunstone (26) for a series of sn-1 monounsaturated/sn-2 monounsaturated C(18:1Delta^n):C(18:1Delta^n)PC. However, for this series of dioctadecenoylphosphatidylcholine, the minimal T(m) value is observed when the Delta bond in both acyl chains lies in between C(9) and C(10).

Before interpreting the V-shaped profile shown in Fig. 2, it is important to mention that all of the DSC heating curves exhibited by aqueous lipid dispersions of C(18):C(18:1Delta^n)PC arecharacterized by a single, sharp, endothermic peak (Fig. 1). This common feature can be reasonably taken as evidence to indicate that each endothermic process is a two-state transition; hence, a simple equilibrium exists between the two phases (gel and liquid-crystalline) at T(m). The T(m) associated with the gel to liquid-crystalline phase transition can thus be expressed by the Clausius equality: T(m) = DeltaH/DeltaS. The variations in calorimetric DeltaH values for aqueous dispersions of C(18):C(18:1Delta^n)PC are small with considerably large errors ( Table 2and Fig. 8B). Nevertheless, these experimental DeltaH values do exhibit a discernible trend characterized by the V-shaped profile over a range of Delta^n from n = 6 to n = 13 (Fig. 8B). On the other hand, the DeltaS values for the same series of C(18):C(18:1Delta^n)PC are essentially constant (Table 1). From this information and the Clausius equality, the V-shaped T(m) profile seen in Fig. 2and Fig. 8A for C(18):C(18:1Delta^n)PC can be simply interpreted by the observed small variations in DeltaH as a function of Delta^n, which is also characterized by a V-shaped profile (Fig. 8B). This DeltaH profile is, by and large, consistent with the (-DeltaE(t)) values calculated for C(18):C(18:1Delta^n)PC (Fig. 8C). Based on the calorimetric and computational data (Fig. 8, A-C), we can conclude that the V-shaped T(m) profile of C(18):C(18:1Delta^n)PC can be explained by a systematic variation in the stability of the C(18):C(18:1Delta^n)PC bilayer, at T < T(m), as a function of Delta^n. One question can immediately be raised: What are the molecular origins of the stability variations for the various gel state bilayers of C(18):C(18:1Delta^n)PC? A plausible answer to this question seems to lie in the energy-minimized structures of C(18):C(18:1Delta^n)PC shown in Fig. 6and Fig. 7, which will be discussed in the following paragraph.

The lipid chain topologies obtained with MM calculations for a series of positional isomers of C(18):C(18:1Delta^n)PC are illustrated graphically in Fig. 6and Fig. 7. These molecular structures show that the sn-2 acyl chain of each of C(18):C(18:1Delta^n)PC molecules is kinked. Comparisons of kinked chains reveal that the two linear segments of the sn-2 acyl chain separated by the Delta-containing kink have different (C-C) values. The values of the longer segments, (C-C), for C(18):C(18:1Delta^n)PC are 10, 9, 7, 6, 7, and 8, respectively, as n = 6, 7, 9, 11, 12, and 13 (Fig. 6). The relative percentage differences among these trans-C-C bond lengths are significant; moreover, the absolute (C-C) value varies in an inverted bell-shaped manner as a function of Delta^n, with the smallest (C-C) value of 6 trans-C-C bond lengths identified for C(18):C(18:1Delta)PC (Fig. 8D). If the longer linear segment is assumed to pack more favorably with its neighboring chains in the two-dimensional plane of the gel state bilayer than does the shorter linear segment, it is then possible to rationalize that the stability of the gel state bilayer will increase with increasing length of the longer segment. Based on the preferential interaction, the DeltaH and hence the T(m) values would be expected to display V-shaped profiles, each with the minimum at Delta. These expectations are of course borne out by calorimetric data ( Table 1and Fig. 2). Thus, we propose that the longer linear segment of the sn-2 acyl chain separated by the Delta-containing kink will tend to pack more effectively with its neighboring chains in the bilayer interior, thereby changing the thermal stability of the bilayer according to the length of the longer segment.

The third important set of results of our DSC studies is depicted in Fig. 4, in which the relationship between the T(m) values of lipid bilayers prepared from different series of monoenoic phosphatidylcholines and the total number of carbon atoms in the sn-1 acyl chains of these lipids is presented. As mentioned under ``Results,'' the calorimetric data shown in Fig. 4can be taken to indicate that the T(m) values of these lipids depend critically on the total number of carbon atoms in both acyl chains as well as the position of the Delta bond along the sn-2 acyl chain. The total number of carbon atoms in the two acyl chains of a phospholipid molecule is related to the thickness of the hydrophobic core of the lipid bilayer in the gel state (N). The effect of the cis-double bond position on the T(m) has just been proposed in the above paragraph to be related to the (C-C) value. Earlier, we mentioned that the chain length difference between the two acyl chains (DeltaC) can perturb the stability of the gel state bilayer. This is an important term for sn-1 saturated/sn-2 monounsaturated phospholipids, since DeltaC remains unchanged for sn-1 monounsaturated/sn-2 monounsaturated phospholipids or when both chains are unsaturated. Next, we want to show that the T(m) values of sn-1 saturated/sn-2 monounsaturated phosphatidylcholines can indeed be correlated quantitatively with their structural characteristics in terms of N, (C-C), and DeltaC.

Before we derive the relationship, it is important to realize that the longer linear segment of the sn-2 acyl chain can be either the upper or the lower linear segment separated by the kink (Fig. 6). The two ends of the upper linear segment are both covalently linked, whereas one end of the lower segment is not. Consequently, the monoenoic lipid can be divided into two groups: group I with a longer upper linear segment, and group II with a longer lower linear segment. For instance, the top two curves in Fig. 4can be identified as lines connecting T(m) values obtained with group I lipids (C(X):C(24:1Delta)PC and C(X):C(22:1Delta)PC), the other three curves connecting T(m) values derived from group II lipids (C(X):C(18:1Delta^6)PC, C(X):C(20:1Delta)PC, and C(X):C(18:1Delta^9)PC). A structural parameter, N(1), can be introduced to specify the importance of the (C-C) values as follows: N(1) = (X - 1) + (C-C) for group I lipids and N(1) = (X - 1) + (C-C) for group II lipids. The values of (C-C) and (C-C) for C(X):C(Y:1Delta^n)PC can be calculated as follows: (C-C) = n - 5 and (C-C) = Y - n - 2. Hence, the structural parameter N(1) specifies the sum of the trans-C-C bond lengths in the sn-1 acyl chain and the longer segment of the sn-2 acyl chain for C(X):C(Y:1Delta^n)PC at T < T(m).

Two structural parameters, N and DeltaC, have been used to correlate with the T(m) value of saturated mixed chain phosphatidylcholines(22) . The parameter N specifies the thickness of the hydrocarbon core of the gel state bilayer, and the other, DeltaC, is the chain length difference between the two acyl chains. For both parameters, the unit is the C-C bond length along the long molecular axis running perpendicular to the bilayer surface. For a saturated mixed chain C(X):C(Y)PC, N and DeltaC are related to X and Y as follows: N = X + Y - 0.5, and DeltaC = X - Y + 1.5(22) . In the case of a monoenoic lipid C(X):C(Y:1Delta^n)PC, the structural parameters N and DeltaC have slightly different expressions: N = X + Y - 1.5, and DeltaC = X - Y + 2.5(23) . Here, the shortening of the sn-2 acyl chain by one C-C bond length caused by the Delta bond has been taken into account in these two expressions. The shortening of one C-C bond length is, in fact, an average value, which can be estimated based on the DeltaC values and their conformational weights (the P value given in Table 3) for all 16 possible kink models of 1-palmitoyl-2-oleoylphosphatidylcholine(21) .

In Table 1, there are 12 molecular species of group I C(X):C(Y:1Delta^n)PC with longer upper segments in the sn-2 acyl chains. The T(m) values of these monoenoic lipids are subjected to multiple regression analysis in an attempt to establish a quantitative T(m)-structure relationship. The statistical analysis is conceptually similar to that established for saturated mixed chain C(X):C(Y)PC(22) , except that an additional structuralparameter, N(1), is introduced for C(X):C(Y:1Delta^n)PC. This analysis yields

with = 0.9973 and the root mean square error = 0.8269, where is the correlation coefficient. The calculated T(m) values based on for all 12 molecular species of group I lipids are given in Table 1. With the only exception as C(20):C(22:1Delta)PC, all of the calculated T(m) values are within ± 0.8 °C of the experiment alones. The calculated T(m) value for C(20):C(22:1Delta)PC, however, is 2.4 °C smaller than the experimentally observed value.

Table 1contains 14 T(m) values determined calorimetrically for group II monoenoic lipids. These values have also been subjected to multiple regression analyses using various combinations of the three structural parameters. gives the best correlation

with = 0.9914 and the root mean square error = 0.9094. The calculated values for the 14 group II lipids are presented in Table 1. With the exception of C(24):C(18:1Delta^9)PC, the differences between the experimental and calculated T(m) values are all within ±1.5 °C. The calculated T(m) value for C(24):C(18:1Delta^9)PC is 1.8 °C smaller than the experimentally observed T(m) value. This largest deviation amounts to only a relative error of 0.62% in absolute temperature.

With the only exception as C(20): C(22:1Delta)PC, the agreement between the calculated and observed T(m) values for sn-1 saturated/sn-2 monounsaturated phosphatidylcholines shown in Table 1is quite reasonable. In the case of C(20):C(22:1Delta)PC, additional DSC experiments with highly purified C(20):C(20:1Delta)PC will be required in the future to clarify the disagreement. Despite this disagreement, it is nevertheless of great significance that there is a commonT(m)-structure relationship that holds for most different monoenoic phosphatidylcholines within each group of sn-1 saturated/sn-2 mono unsaturated phosphatidylcholines. This common relationship suggests strongly that the various parameters characterizing the fundamental structures of monoenoic lipids at T <T(m) also contribute collectively to the unique T(m) values of these lipids packed in the bilayer.


FOOTNOTES

*
This investigation was supported in part by United States 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. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
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.

(^1)
The abbreviations used are: DSC, differential scanning calorimetry; MM, molecular mechanics; T, phase transition temperature; DeltaH, transition enthalpy; E, the steric energy, in kcal/mol, of an energy-minimized lipid species; C(X):C(Y)PC, saturated phosphatidylcholine or 1,2-diacyl-sn-glycero-3-phosphocholine with X carbons in the sn-1 and Y carbons in the sn-2 acyl chain; C(X):C(Y:1Delta)PC, monounsaturated phosphatidylcholine with a saturated sn-1 acyl chain containing X carbons and a monounsaturated sn-2 acyl chain containing Y carbons with a cis-carbon-carbon double bond (Delta) at the nth carbon atom from the carboxyl end; DeltaT, transition peak width at half-height; PE, phosphatidylethanolamine; DeltaS, phase transition entropy change; DeltaH, weight average transition enthalpy; F-B, front to back; U-D, up to down.


ACKNOWLEDGEMENTS

We thank Professor T. E. Thompson for critical comments on the manuscript.


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