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INTRODUCTION |
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 carbon-carbon double
bonds are invariably separated by a three-carbon unit comprised of a
methylene group (-CH2-) sandwiched by two olefinic
carbons. The biochemical significance of this regular
methylene-interrupted interval is, however, not clear.
Although it has long been known that mixed-chain diacyl phospholipids
in aqueous media can uniquely assemble into a two-dimensional sheet-like structure called the lipid bilayer, progress is nonetheless slow in understanding how variations in the chemical composition of
fatty acyl chains affect the structure/property relationship of the
lipid bilayer. This is, in part, due to the fact that up to the present
time not a single x-ray crystal structure of mixed-chain phospholipid
is available. A second reason is that many diacyl mixed-chain
phospholipids containing single or multiple cis double bonds
are rather difficult to synthesize (or semi-synthesize) in a typical
biochemical or biophysical laboratory. Nevertheless, the pioneer work
of Keough and co-workers (1-3) did provide interesting results showing
how variations in the number of cis double bonds in the
sn-2 acyl chain of phosphatidylcholine (PC) at certain fixed
positions can affect the phase transition temperature
(Tm) of the lipid bilayer. Specifically, Keough and
associates (1-3) have shown by DSC that the introduction of a
cis double bond into the sn-2 acyl chain of
C(20):C(20)PC at carbon 11 from the carbonyl end, or
C(11), gives rise to
C(20):C(20:1
11)PC, which has a considerably lower
Tm relative to its saturated counterpart. The
introduction of a second cis double bond into the
sn-2 acyl chain at the methylene-interrupted position toward
the methyl end yields C(20):C(20:2
11,14)PC with a
further reduction in Tm. Interestingly, the introduction of a third cis double bond at C(17)
results in a small increase in Tm for
C(20):C(20:3
11,14,17)PC. This down and up trend in
Tm has been confirmed calorimetrically by other
groups (4, 5). One can immediately raise a relevant question as to
whether this down and up Tm profile is a special or
a general characteristic for acyl chain unsaturation. Phrased
differently, what kind of Tm profile will be
observed if the first
-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 Tm 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 < Tm have to be estimated.
Recently, we have semi-synthesized a limited number of diacyl
mixed-chain phosphatidylethanolamines (PE) with sn-1
C20-saturated and sn-2
C20-unsaturated acyl chains (6-9). Our calorimetric data
showed that the Tm profile exhibited by a series of
mixed-chain PE containing 1-3 cis
-bonds in the
sn-2 C20-acyl chains at
11-,
11,14-, and
11,14,17-positions,
respectively, is parallel to that displayed by the corresponding
mixed-chain PC observed earlier by Keough et al. (8). The
influence of single and multiple
-bonds on the chain-melting behavior of PC and PE bilayers thus appears very similar. If we can
delineate the common structural features governing the shapes of
Tm profiles for various series of mixed-chain PE, the information obtained may provide an understanding of the
structure/property relationships underlying most other naturally
occurring phospholipids. With this in mind, in the present study we
have extended the list of synthesized mixed-chain PEs containing
sn-1 C20-saturated and sn-2
C20-unsaturated acyl chains to a total of 15 different
species. The phase transition behavior of these mixed-chain PE, in
excess H2O, has also been investigated by high resolution
DSC. Based on the calorimetric data, a novel Tm
diagram is generated for the first time. In this diagram,
Tm values of 9 series of mixed-chain PE each
containing three or more lipids are systematically arranged. The shapes
of Tm profiles displayed by various lipids in all
nine series can be seen simultaneously in plots derived from the
Tm diagram. Furthermore, in this study we have used
the computer-based molecular mechanics (MM) calculations to simulate
the energy-minimum structures of these mixed-chain PEs. The
characteristic Tm profile obtained with lipids in
each series of mixed-chain PE can be interpreted in terms of structural
changes of the sn-2 acyl chain of the lipid resulting from
acyl chain unsaturation.
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EXPERIMENTAL PROCEDURES |
Chemicals--
With the exception of six species of unsaturated
C20 fatty acids that were synthesized in this laboratory as
described in the next paragraph, all other C20-unsaturated
fatty acids including arachidonic acid were obtained from Sigma.
Lysophosphatidylcholine with a C20-acyl chain was purchased
from Avanti Polar Lipids (Alabaster, AL). Phospholipase D, type I from
cabbage, was obtained from Sigma. Chemicals used for the fatty acid
synthesis were supplied by Aldrich. All routine reagents and organic
solvents were of reagent and spectroscopic grades, respectively, and
they were obtained from various commercial sources.
Synthesis of Unsaturated C20 Fatty Acids and
Semi-synthesis of Mixed-chain PE--
In the present investigation,
six species of C20-unsaturated fatty acids were
synthesized; they were cis-14-eicosenoic,
cis-17-eicosenoic, cis,cis-5,8-eicosadienoic,
cis,cis-8,11-eicosadienoic,
cis,cis-14,17-eicosadienoic, and
all-cis-8,11,14,17-eicosatetraenoic acids. For monoenoic
acids, the synthesis was carried out based on the method of Holman and co-workers (10, 11). By using the synthesis of
cis-14-eicosenoic acid as an example, this method can be
briefly described as follows: the starting material is
1-bromoundecan-11-ol. After the hydroxyl group has been protected by
3,4-dihydro-2H-pyran, the primary alkyl derivative can
interact with heptyne-1 in the presence of butyl lithium to yield
-hydroxyl alkyne. Upon further reacting with
CH2(CO2Et)2 in the presence of
EtONa, the chain elongation step gives a product of appropriate total
number of carbons, viz. the eicosa-14-ynoic acid. Finally,
the triple bond of this C20-alkynoic acid is
cis-hydrogenated using Lindlar catalyst to form
cis-14-eicosenoic acid. The syntheses of various dienoic
C20 fatty acids were accomplished by the established
procedure published recently from this laboratory (9). For the
synthesis of all-cis-8,11,14,17-eicosatetraenoic acid, the
method of Osbond et al. (12) was employed, in which 1-bromoundeca-2,5,8-triyne and 8-nonyoic acid were coupled to form
8,11,14,17-eicosatetraynoic acid followed by
cis-hydrogenation to form the final product of
all-cis-tetraenoic acid. The purity of the synthesized mono-
and dienoic acids was estimated by high pressure liquid chromatography.
Mixed-chain sn-1 C20-saturated/sn-2
C20-unsaturated PE was semi-synthesized from the
corresponding PC by the base-exchange reaction in the presence of
excessive amounts of ethanolamine hydrochloride, at pH 5.6, using
phospholipase D according to the method of Comfurius and Zwaal (14) as
described in detail elsewhere (6, 8). The semi-synthesis of mixed-chain
PC involved the acylation of C(20)-lysophosphatidylcholine
with C20 fatty acid in the presence of catalyst
4-pyrrolidinopyridine using the established procedure reported
previously (15). In semi-synthesizing mixed-chain PE, all chemical and
enzymatic reactions were carried out strictly under N2 to
avoid possible lipid oxidation. All lipids were purified by column
chromatography on silica gel 60, with which a mixture of
CHCl3, CH3OH, 5% NH4OH, 175:35:4
(v/v/v) was used as the eluant. Only a single spot was observed for
each of the PE synthesized, after about 1 µmol per sample was loaded
on the thin layer plate and developed in CHCl3,
CH3OH, 5% NH4OH (65:30:5). Prior to use, the
lipid powder obtained from lyophilization of the lipid/benzene solution
was kept at
20 °C.
High Resolution DSC Measurements--
The lipid samples used for
DSC experiments were prepared according to our previously reported
protocols (9). Specifically, the lyophilized lipid power was dispersed
in cold aqueous buffer solution containing 50 mM NaCl, 0.25 mM diethylenetriaminepentaacetic acid, 5 mM
phosphate buffer, pH 7.4, and 0.02 mg/ml NaN3. All DSC
experiments were performed on a MicroCal MC-2 calorimeter with a DA-2
digital interface and data acquisition utility for automatic collection
(Microcal, Northampton, MA). In these DSC runs, a constant heating scan
rate of 15 °C/h was used; lipid samples were scanned a minimum of
three times with at least 60-90 min of equilibration at low
temperatures between scans. As in our previous studies (6-9), the
phase transition temperature and the transition enthalpy were obtained
from the second DSC heating curve. Specifically, the gel-to-liquid
crystalline phase transition temperature, Tm,
corresponds to the peak position with maximal peak height, and the
transition enthalpy,
H, can be determined from the area
under the transition peak and the lipid concentration using the
software provided by Microcal. In general, the Tm
value obtained at the transition with maximal peak height from the
second DSC heating run was reproduced at ± 0.1 °C for each
lipid sample. The
H value, however, has a considerable
higher error owing to the uncertainty in deciding the onset and
completion temperatures of the transition curve. The relative errors
may amount to 20% for very broad transition curves as exhibited by
some polyunsaturated lipids.
Molecular Mechanics (MM) Calculations--
All molecular
mechanics (MM) force field calculations were carried out using an IBM
RS/6000 computer workstation. The software MM3 (version 92) for MM
calculations was supplied by Quantum Chemistry Program Exchange,
Chemistry Department, Indiana University, Bloomington, IN. The MM3
computation began with the input of the estimated atomic coordinates
for mixed-chain PE followed by systematic adjustment of the structural
parameters by repeated automatic cycles of the Newton-Raphson
minimization technique (16). These cycles of self-adjusted computation
came to a halt as the steric energy reached the minimum. The structural
data resulting from the MM3 computation were stored. Subsequently,
these data were transferred into a Pentium P5-200 platform equipped
with HyperChem 4.0 software (HyperCube, Gainesville, FL), from which
the three-dimensional graphic images of the energy-minimized lipid
molecules can be visualized. Details of the procedure for obtaining the
energy-minimized structure for each sn-1
saturated/sn-2 unsaturated phospholipid were described
previously (17-19). It should be mentioned, however, that prior to
stochastic search for the energy-minimized conformation of
sn-1 saturated/sn-2 unsaturated PE, the atomic
coordinates (e.g. torsion angles) of the initially crude
structure for a given mixed-chain PE were estimated based on the single
crystal structure of C(12):C(12)PE (20) and the energy-minimized
unsaturated fatty acyl chain (18). Any additional methylene-interrupted
cis double bonds for the sn-2 acyl chain were
constructed by using
s
s+s+
s
(or
s+
s
s
s+)
as the added sequence (18), where s± refers to
skew (±) conformations with torsion angles of about ± 110o and
denotes cis double bond with
torsion angle of about 0o. These initial coordinates of the
crude structural model were used as a set of initial input data in MM3 computations.
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RESULTS |
The Phase Transition Behavior of Lipid Bilayers Composed of PE with
sn-1 Saturated C20 and sn-2 Unsaturated
3(or
6)C20 Acyl Chains--
Fig.
1 shows the second DSC heating curves for
aqueous dispersions prepared individually from a saturated identical
chain C(20):C(20)PE and its five unsaturated
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
3-position) 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, Tm, as reported previously (21). After
the first cis carbon-carbon double bond is introduced into
the sn-2 acyl chain of C(20):C(20)PE at the
3 (or
17)-position, the resulting C
(20):C(20:1
17)PE also displays a single, sharp
endothermic transition; however, its Tm decreases by
15.7 °C, relative to its saturated counterpart, from 82.5 to
66.8 °C (Fig. 1). This single endothermic transition can also be
attributed to the gel-to-liquid crystalline phase transition.
Furthermore, as a cis double bond is introduced successively
at a regular methylene-interrupted interval toward the carbonyl end,
various polyunsaturated PEs with increasing numbers of double bonds are
formed. These polyunsaturated PEs, in excess water, display
calorimetrically broad gel-to-liquid crystalline phase transitions each
with a characteristic Tm (Fig. 1). Interestingly, in
the plot of Tm versus the number of
double bonds as illustrated in the inset of Fig. 1, the
Tm value is observed to decrease nonlinearly with a
stepwise increase in the number of cis carbon-carbon double bonds in the sn-2 acyl chain. In particular, the
Tm increment is diminished substantially between
C(20):C(20:3
11,14,17)PE and
C(20):C(20:5
5,8,11,14,17)PE. This nonlinear decrease in
Tm reflects that the successive increase
in the degree of acyl chain unsaturation has a nonadditive
Tm lowering effect. Nevertheless, all experimental Tm values fit reasonably well by a least squares
binomial curve with a correlation coefficient of 0.9836 (the
inset of Fig. 1). Values of Tm,
H, and other thermodynamic parameters associated with the
gel-to-liquid crystalline (or chain-melting) phase transition for all
five species of unsaturated
3 derivatives of C(20):C(20)PE are
summarized in Table I.

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Fig. 1.
The representative second DSC heating curves
for aqueous dispersions prepared individually from C(20):C(20)PE and
its five unsaturated 3 derivatives. These
unsaturated 3PEs contain 1-5 cis -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
3- or 17-position). The abbreviated name for each
unsaturated lipid species is indicated under each transition curve, and
above the transition curve, the value of Tm obtained
with each of the six lipid dispersions is also indicated. Furthermore,
the Tm value obtained with each individual 3PE is
plotted in the inset against the number of cis
-bonds in that lipid species.
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Table I
The thermodynamic parameters associated with the chain-melting
phase transitions of C(20):C(20)PE and its unsaturated 3 and
6 derivatives
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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 Tm value. Moreover,
a nonlinearly decreased Tm curve in the plot of
Tm versus the number of cis
carbon-carbon double bonds is observed (the inset of Fig. 2). In particular, the lipid species with an sn-2
arachidonyl (or all-cis-5,8,11,14-eicosatetraenoyl) chain
has the smallest Tm value of 6.6 °C. It should be
mentioned that the phase transition behavior of C(20):C(20)PE and some
of its
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 C20-saturated/sn-2
C20-
6-unsaturated acyl chains. In Table I, the values of
Tm,
H, and other thermodynamic parameters associated with the phase transition of C(20):C(20)PE and
its four
6-unsaturated derivatives are summarized.

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Fig. 2.
The representative second DSC heating curves
for aqueous dispersions prepared individually from a saturated
identical chain C(20):C(20)PE and its four unsaturated
6 derivatives. These unsaturated 6PEs
contain 1-4 cis -bonds in the sn-2
acyl chain of the lipid, with the position of the commonly shared
double bond being 6 carbons from the methyl end (the 6- or
14-position). The phase transition temperature,
Tm, for each lipid dispersion is plotted in the
inset against the number of cis
-bonds in the sn-2 acyl chain of the
corresponding 6PE.
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As described under "Experimental Procedures," the monounsaturated
C(20):C(20:1
14)PE was semi-synthesized by acylation of
C(20)-lyso-PC with cis-14-eicosenoic acid followed by
base-exchange reaction in the presence of excessive ethanolamine.
Furthermore, the monoenoic
6 fatty acid
(cis-14-eicosenoic acid) was also synthesized in this
laboratory. The single and symmetric endothermic transition with
Tm = 47.4 °C and
T1/2 = 0.9 °C exhibited by the C(20):C(20:1
14)PE dispersion
as seen in Fig. 2 can be taken as strong evidence indicating that the
monounsaturated
6PE and the monoenoic
6 fatty acid synthesized in
this laboratory have high degrees of isomeric and chemical purities.
Furthermore, the single phase transition at 16.4 °C exhibited by the
aqueous dispersion of C(20):C(20:4
8,11,14,17)PE is seen
in Fig. 1 to be symmetrical with a
T1/2 of
2.4 °C, reflecting again the high degree of purity of our
synthesized all-cis-8,11,14,17-eicosatetraenoic acid as well
as the synthesized C(20):C(20:4
8,11,14,17)PE.
The Tm Diagram for PE with sn-1
C20-saturated/sn-2 C20-unsaturated Acyl
Chains--
In recent years, the phase transition behavior of six
lipid species of C(20):C(20)PE derivatives with
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 Tm values from these six
PEs and those obtained with
3 and
6 PEs shown in Figs. 1 and 2
are codified, a general Tm diagram for
sn-1 C20-saturated/sn-2
C20-unsaturated PE can be generated for the first time
(Fig. 3). Specifically, the
Tm diagram shown in Fig. 3 has the shape of a
right-angled triangle, comprising 15 species of sn-1
C20-saturated/sn-2 C20-unsaturated PE. These lipids are arranged into 5 levels depending on the position of the
-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 Tm value of any given
sn-1 C20-saturated/sn-2
C20-unsaturated PE is shown under the abbreviated name of
the given lipid species in Fig. 3. Vertically, each column in the
Tm diagram also represents a series of unsaturated
PEs, which share a common
n-bond. Hence, each series is
designated as the
nPE 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 Tm
diagram, the effects of acyl chain mono- and polyunsaturation on the
chain-melting behavior of lipid bilayers can be examined directly in a
unifying manner.

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Fig. 3.
The Tm diagram for
sn-1 C20-saturated/sn-2
C20-unsaturated PE. Lipids in each row share a common
-carbon, where the -carbon is defined as the first olefinic
carbon atom in the sn-2 acyl chain of the lipid when
counting from the chain terminal methyl end. The five parallel rows of
unsaturated lipids from top to bottom are
15PE, 12PE, 9PE, 6PE, and 3PE series as indicated.
Lipids in each column share a common n-bond, and lipids in
each column thus belong to a nPE series, where the
superscript n denotes the position of the common
cis carbon-carbon double bond in the sn-2 acyl
chain when counting from the carbonyl end. In this case, the carbonyl
carbon is designated as the first carbon of the acyl chain. The five
columns of unsaturated lipids from left to right
are 17PE, 14PE, 11PE,
8PE, and 5PE series, respectively, as
indicated. All together, there are 15 molecular species of unsaturated
mixed-chain PE in this Tm diagram, and their
Tm values are given under the abbreviated names of
the corresponding mixed-chain PE.
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For lipid species aligned horizontally along each row in the
Tm diagram (Fig. 3), the added cis
carbon-carbon double bond is introduced at a regular
methylene-interrupted interval, proceeding toward the carbonyl end of
the sn-2 acyl chain. In addition, this Tm
diagram shows another common feature exhibited by each series of the
(3-12)PE as follows. The Tm decreases
continuously but nonlinearly with a stepwise increase in the number of
cis double bonds. Consequently, we can arrive at a general
conclusion that the gel-to-liquid crystalline phase transition behavior
of PE bilayers is influenced markedly in a systematic way by the number
of cis carbon-carbon double bonds present in the
sn-2 acyl chain of the PE.
The lipid species aligned vertically along each column in the
Tm diagram also show a growing number of
cis double bonds in the sn-2 acyl chain of the
lipid (Fig. 3). However, the methylene-interrupted cis
double bond is added on the methyl side of the existing double bond.
Interestingly, the Tm values exhibited by the
8-PE series of C(20):C(20:1
8)PE,
C(20):C(20:2
8,11)PE,
C(20):C(20:3
8,11,14)PE, and
C(20):C(20:4
8,11,14,17)PE and the
11-PE
series of C(20):C(20:1
11PE,
C(20):C(20:2
11,14)PE, and
C(20):C(20:3
11,14,17)PE show down and up trends, which
can be viewed as an exception of the continuously downward trend
of Tm normally observed with most lipid series that
contain a stepwise increase in the number of cis double
bonds. A similar down and up Tm trend has been
detected earlier for a
11-PC series of
C(20):C(20:1
11PC, C(20):C(20:2
11,14)PC,
and C(20):C(20:3
11,14,17)PC by Keough et al.
(1, 3).
The Tm diagram shown in Fig. 3 can also be viewed
diagonally. For the monoenoic series of PE, the lipid with the cis double bond located in the middle of the sn-2
acyl chain, C(20):C(20:
11)PE, has the lowest
Tm value of 43.3 °C; moreover, the Tm value increases as the single cis bond
migrates from the middle toward the carbonyl or methyl end (Fig. 3),
thereby resulting in a roughly V-shaped Tm profile.
Similar Tm profiles can also be recognized for
dienoic and trienoic series of PE as seen diagonally in the
Tm diagram (Fig. 3). Based on these diagonal terms,
one can conclude that for lipids with a fixed number of cis
double bonds, the position of the double bonds along the acyl chain of
the lipid can notably affect the chain-melting behavior. It should be
mentioned that various V-shaped Tm profiles for the
monoenoic, dienoic, and trienoic series of PE shown in the plot of
Tm versus the position of cis
double bonds have been documented previously, albeit separately, in the
literature (6, 8, 9). Uniquely, the Tm diagram
illustrated in Fig. 3 shows simultaneously all these
Tm profiles in a simple and unifying manner.
The most important conclusion that can be drawn from the
Tm diagram illustrated in Fig. 3 is that the number
and the position of cis double bonds in the sn-2
acyl chain of PE can characteristically influence the gel-to-liquid
crystalline phase transition temperature of the PE bilayer. To
recapitulate this important point further, all Tm
values of C(20):C(20)PE derivatives shown in Fig. 3 are plotted
three-dimensionally against the number and the position of
cis double bonds. Specifically, in the plot of Fig.
4A,
(n)-carbon
represents the first olefinic carbon at the position of n
from the methyl end that is shared by all lipids in a series of
unsaturated PE. It is evident that all Tm values
within each series of the
(3-12)PE as depicted in Fig.
4A exhibit a common feature, a continuously downward shift
in Tm as the degree of chain unsaturation increases successively. In addition, roughly V-shaped Tm
profiles are shared by mono-, di-, and trienoic series of PE as the
position of cis double bonds migrates from the
3-carbon
to the
15-,
12-, and
9-carbons, respectively, in the
sn-2 acyl chain. In Fig. 4B, the position
of the commonly shared cis bond is represented by the
n-carbon, where the superscript n denotes the
position of the first olefinic carbon in the sn-2 acyl chain
when counting from the carbonyl end. Discernibly, for lipids in the
8-PE or
11-PE series the variation of
Tm is characterized by a down and up trend.

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Fig. 4.
Tm values of unsaturated
mixed-chain PE shown in Fig. 3 are plotted
three-dimensionally against the number and the position of
cis double bonds. In the plot of A,
(n)-carbon represents the first olefinic carbon at the
position of n from the methyl end. B, the
position of the commonly shared cis -bonds is
n-carbon, where the superscript n denotes the
position of the first olefinic carbon in the sn-2 acyl chain
when counting from the carbonyl end.
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The Energy Minimized Structures of sn-1
C20-saturated/sn-2 C20-unsaturated PE as
Determined by Molecular Mechanics Simulations--
X-ray
crystallographic structures of diacyl phospholipids with
sn-1 saturated and sn-2 unsaturated chains have
not been determined at the present time. However, based on the atomic
coordinates of the single crystal structures of saturated diacyl
phospholipids and unsaturated fatty acids, the energy-minimized
structures of some unsaturated phospholipids in the crystalline state
bilayer have been simulated by molecular mechanics (MM) calculations
(18, 19). This MM approach is employed in the present study to
construct the minimum energy structures for various PEs with
sn-1 C20-saturated/sn-2 C20-unsaturated acyl chains.
In Fig. 5, the energy-minimized
structures of six lipid species in the
3PE series are presented
using space-filling and wire models. These structures can be taken to
approximate the monomeric lipids packed in the crystalline bilayer.
Here, the head groups of all six lipid molecules are aligned
identically. Furthermore, the zigzag plane of the all-trans
sn-1 acyl chain of each individual lipid species is seen in the
wire model to lie perpendicularly to the paper plane, whereas the
sn-2 acyl chain is seen in the space-filling model to
project in front of the sn-1 acyl chain. For C(20):C(20)PE,
the segment of the sn-2 acyl chain running approximately in
parallel with the all-trans sn-1 acyl chain extends from
C(3) to C(20) with 17 C-C bond lengths. However,
the all-trans segment (ATS) of the sn-2 acyl
chain is assumed to extend from C(3) to C(19)
with 17 methylene units (Fig. 5). This assumption is based on the
notion that the chain terminal CH2-CH3 bond is usually disordered at T < Tm,
particularly for lipids packed in the gel state bilayer. In the case of
monounsaturated C(20):C(20:1
17)PE, the sequence of the
17-containing kink in the sn-2 acyl chain is
s
s
, where
s
and
are skew(
) and
cis double bonds, respectively. By MM calculations, a set of
optimal torsion angles for this
s
s
sequence is
determined to be (
109°,
1.1°, and
120°) as indicated in
Fig. 5. Consequently, the sn-2 acyl chain of
C(20):C(20:1
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 C
C single bond preceding the
17-bond has a
skew(
) conformation with an optimal torsion angle of
109°. 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± 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.

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Fig. 5.
The energy-minimized structures of
identical-chain C(20):C(20)PE and its five unsaturated
3 derivatives as shown molecular graphically by
space-filling and wire models. These unsaturated 3PEs contain
1-5 cis -bonds. The ATS shown in the wire model denote
the all-trans segment of the hydrocarbon chain in the
sn-2 acyl chain. The kink sequence in the
sn-2 unsaturated acyl chain and a set of optimal
torsion angles associated with the kink sequence are presented
below each unsaturated lipid species. It should be noted
that the length of ATS is shortened progressively as the new
methylene-interrupted cis-double bond is added successively
on the carbonyl side of the 17-bond in the
sn-2 acyl chain of C(20):C(20:1 17)PE.
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The energy-minimized structures of the five lipids listed in the
right column in the Tm diagram (Fig. 3)
have also been determined by MM calculations using the MM3 program
(data not shown). Like lipids in the
3PE series shown in Fig. 5,
lipids in this
5PE series contain up to 5 methylene-interrupted cis carbon-carbon double bonds in the
sn-2 acyl chain. The chemical and molecular structures of
these lipids, however, differ from those in the
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 C
C bond
lengths, extending from C(3) to C(5) for all
lipids in this series. In contrast, the length of ATS is shortened
progressively as the new cis double bond is added
successively into the sn-2 acyl chain. This decreasing trend
in ATS is, in essence, identical to that observed in Fig. 5 for the
3 series of C(20):C(20)PE.
The minimum-energy structures of C(20):C(20:1
11)PE,
C(20):C(20:2
11,14)PE, and
(20):C(20:3
11,14,17)PE in the
11PE series
are illustrated in Fig. 6 by
space-filling and wire models. For C(20):C(20:1
11)PE,
the
11-double bond is seen to be in the middle of the
sn-2 acyl chain, and the additional
-bonds are on the
methyl side of the
11-bond. Unlike the variable
length of ATS shown in Fig. 5, all three unsaturated lipid species in
this series of
11PE share a constant chain length of ATS
extending from C(3) to C(10) in the sn-2 acyl chain.

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Fig. 6.
Molecular graphics representations of the
energy-minimized structures of
C(20):C(20:1 11)PE,
C(20):C(20:2 11,14)PE, and
(20):C(20:3 11,14,17)PE in the
11PE series as depicted by
space-filling and wire models. It should be noted that ATS in each
lipid species is located in the upper segment of the kinked
sn-2 acyl chain, and the added -bond is introduced on the
methyl side of the existing -bond. Here, the ATS is defined as the
all-trans segment of consecutive -CH2- groups
located in the long segment of the kinked sn-2 acyl chain.
The kink sequence and associated torsion angles for each unsaturated
acyl chain are presented under the energy-minimized structure of the
corresponding lipid. These torsion angles were obtained with the MM3
program as described under "Experimental Procedures."
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|
In Fig. 7, the energy-minimized
structures of lipids in the
8PE series are illustrated
using space-filling and wire models. These four; not five lipid species
share a common segment of 5 consecutive methylene units, extending from
C(3) to C(7), in the sn-2 acyl chain. For C(20):C(20:2
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.

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Fig. 7.
The energy-minimized structures of lipids in
the 8PE series as represented by
space-filling and wire models. Initially, the ATS is located in
the lower segment of the unsaturated sn-2 acyl chain. In
accompanying the C(20):C(20:2 8,11)PE (20):C(20:3 8,11,14)PE conversion, the ATS is seen to
shift from the lower segment to the upper segment. Here, ATS is defined
as the all-trans segment of consecutive -CH2-
groups located in the long segment of the kinked sn-2 acyl
chain. The torsion angles of the kink sequence in each lipid species
are given under the two structural models of the appropriate lipid
molecule.
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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 < Tm. Unlike molecular dynamics simulations, the
MM-simulated structure does not explicitly provide information about
the dynamic nature of lipid molecule. For instance, the sn-2
acyl chains of all unsaturated
3PEs share a common chain terminal
segment of
C(16)
C(17)=C(18)
C(19)
C(20), in which the C
C single bonds are all rotationally highly dynamic at
T < Tm. Hence, this disordered
methyl-terminal segment does not undergo the thermally induced
trans
gauche isomerizations at Tm.
The dynamic nature of this short terminal segment is, however, not
revealed by the MM-simulated structure. On the other hand, as the
number of
-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
Tm in the rest of the "Discussion," are clearly indicated in the MM-simulated structures as depicted in Fig. 5.
When a cis carbon-carbon double bond (
) is introduced
into a long hydrocarbon chain, the six atoms in the immediate vicinity of the
-bond, C
CH=CH
C, are coplanar. Although the rotational flexibility of the
-bond in the six-atom unit is highly restricted at physiological temperatures, paradoxically the C
C single bond preceding or succeeding the
-bond is rotationally highly flexible, in terms of torsion-angle fluctuations, even at very low temperature of
10 °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 < Tm can be reasonably assumed to be highly
disordered, and hence it contains virtually no trans
rotamers. We believe that this assumption is justified by its utility
in the following discussion.
Fundamentally, the thermally induced gel-to-liquid crystalline phase
transition of the lipid bilayer occurring at Tm involves principally the trans
gauche rotational
isomerizations of methylene groups about C
C single bonds along the
acyl chains of the lipid (22). Since the short segment of the kinked
sn-2 acyl chain is assumed highly disordered in the
gel-state bilayer at T < Tm, it
thus makes no contributions to the chain disordering process at
Tm. However, consecutive methylene groups in both
the ATS of the sn-2 acyl chain and the all-trans sn-1 acyl chain can be induced thermally to undergo the
disordering process of trans
gauche isomerizations. When
we compare the thermodynamic parameters (Tm,
H, and
S) associated with the chain-melting
phase transition for unsaturated lipids in the
3PE series at
T < Tm, we mention primarily the length of ATS. This is due to the fact that in the gel-state bilayer an
identical length of the all-trans sn-1 C20-acyl
chain exists in all lipids in the
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
C20-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 = Hlc
Hgel, where
H is the enthalpy of the lipid bilayer and the subscripts lc
and gel denote the liquid-crystalline and gel phases of the lipid
bilayer, respectively. It is well known that saturated PE and its
sn-1 saturated/sn-2 unsaturated derivatives are
highly dynamic and disordered in lipid bilayers in the
liquid-crystalline state; hence, the lateral chain-chain contact
interactions are minimal among lipids in these liquid-crystalline
bilayers. We can thus assume that Hlc is
virtually identical for lipids with 0-5
-bonds. As a result, the
H trend exhibited by lipids in the
3PE series can, to
a first approximation, be related to the Hgel
values for these lipids. For unsaturated lipids packed in the ordered
gel-state bilayer, the lateral chain-chain van der Waals attractive
interactions can be directly related to the length of ATS; moreover,
these interactions are also modulated by the dynamic state of the ATS.
As the first two cis C-C double bonds are introduced into
the sn-2 acyl chain of C(20):C(20)PE at the
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
Hgel 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 Hgel. 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 Hgel and hence
H values are
nearly identical.
Similarly, the transition entropy for the chain-melting phase
transition can be expressed as
S = Slc
Sgel. For lipids
in the
3PE series, the value of Slc 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 Sgel
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
Sgel value increases markedly due to the high
flexibility of C-C single bonds adjacent to the
-bonds. Above 2, the Sgel decreases with increasing
-bonds as a result of the increased rigidity of multiple
-bonds. In
particular, the maximal Sgel 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
Sgel. Based on the proposed linear
Slc curve and the nonlinear
Sgel 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 Tm
as a function of alterations in the number of
-bonds can now be considered. First, the following identity holds: Tm=
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 Tm profile observed in Fig. 1,
we can conclude that the origin of Tm is largely
enthalpic, not entropic. Hence, for each lipid in the
3PE series the
main contribution to Tm is the overall lateral
chain-chain attractive van der Walls interaction in the gel-state
bilayer. However, in view of all lipids in the
3PE series,
particularly those with 3-5
-bonds, the relative
Tm values must also be modulated differentially by
entropic variations. As a result, the shape of the
Tm profile varies somewhat from that of the
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 Tm is not
possible. Nevertheless, the relative magnitudes of
Tm 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 Tm profile seen in Fig. 1 (the inset) can
be reasonably attributed to the net result of the following two
opposing effects as follows: 1) the Tm lowering
effect caused by the progressive shortening of ATS, and 2) the
Tm elevating effect exerted by the increasing
rigidity of 3-5
-bonds. It is important to point out that the
shortening of a three-carbon interval in the ATS has a more pronounced
Tm lowering effect than the opposing effect of the
added
-bonds for lipids in this series of
3PE. Consequently, the
Tm profile seen in the inset of Fig. 1 is
characterized by a decreasing, not an increasing, temperature mode. It
is perhaps worth mentioning that the two opposing effects are caused
paradoxically by the same structural change, viz. the
increasing degree of acyl chain unsaturation in the sn-2
acyl chain.
We further postulate that the combined effects of the length of ATS and
the multiple
-bonds on Tm 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 Tm 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
Tm. However, the magnitude of the
Tm 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 Tm, but this
Tm elevating effect is less than the
Tm lowering effect exerted by the shortening of the
ATS in the sn-2 acyl chain. On balance, the
Tm values in the plot of Tm
versus the number of
-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
11PE series as
shown in the Tm 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
s
g
.
Here, the upper segment designates the chain segment closer to the
bilayer/H2O interface, and the lower segment is assigned to
the one containing the methyl group. In the kinked sn-2
cis-11-eicosenoyl chain of C(20):C(20:1
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
11PE series are obtained if the incorporation of
-bonds takes place in the upper and lower segments of
cis-11-eicosenoyl chain, respectively. Interestingly, the
Tm profiles exhibited by these two series of
unsaturated PE are distinctly different (Fig. 4, A and B), indicating that the position of the incorporated
-bond can markedly affect the chain-melting behavior. The shape of
each of the two Tm profiles will be interpreted
later in terms of the length and location of ATS and the rigidity of
the multiple
-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
gauche isomerizations in comparison with the equivalent length
of ATS located in the lower chain segment. Hence, when two lipids with
the same length of ATS are compared, the one with ATS in the upper
segment will have a higher Tm. Similarly, if a short
segment of consecutive methylene units is in the upper portion of the
sn-2 acyl chain and its length differs from that of ATS in
the lower segment by only one or two -CH2- units, then
this short segment can most likely undergo the thermally induced
trans
gauche isomerizations. It thus contributes to the
overall chain-melting process. With these basic concepts in mind, we
can rationalize the characteristic Tm profile exhibited by lipids in the
9PE series.
The three lipids in the
9PE series,
C(20):C(20:1
11)PE, C(20):C(20:2
8,11)PE,
and C(20):C(20:3
5,8,11)PE, share a common
lower segment of 7 consecutive methylene units in their sn-2
acyl chains. As shown in Figs. 6I and 7II, the
C(20):C(20:1
11)PE
C(20):C(20:2
8,11)PE
conversion is accompanied by two distinctive structural changes as
follows: the length of ATS is shortened by one -CH2- unit, and the position of ATS is shifted from the upper to the lower chain
segment. Both changes can result in a lowering of
Tm. Indeed a decrease of 13 °C in
Tm is observed experimentally to accompany such a
conversion (Fig. 3). The third member of the
9PE series is the
trienoic C(20):C(20:3
5,8,11)PE. Although the
MM-simulated structure is not shown, this trienoic lipid contains an
ATS that is identical to the corresponding ATS in the dienoic
C(20):C(20:2
8,11)PE. Despite the identical position and
length of ATS, the Tm of
C(20):C(20:3
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
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
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 Tm. However,
all-cis-5,8,11-eicosatrienoyl chain has three
-bonds, which can elevate the Tm somewhat by retarding
sterically the trans
gauche isomerizations of
neighboring chains in bilayers. As a result, the
Tm between C(20):C(20:2
8,11)PE and
C(20):C(20:3
5,8,11)PE is smaller than that between
C(20):C(20:1
11)PE and
C(20):C(20:2
8,11)PE.
Thus far, we have discussed four Tm profiles
exhibited by lipids from
3-,
6-,
9-, and
5PE
series. All four Tm profiles show a similar pattern; the Tm 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
11PE series; interestingly, the
Tm profile displayed by lipids from each of the
8- and
11PE series is characterized by a
down and up pattern as illustrated in Fig. 4B. Each of the
down and up Tm profiles will be analyzed in the
subsequent section; in particular, the structural features of those
lipid species that give rise to higher Tm upon
further unsaturation will be delineated. Let us begin with the
11PE series.
The energy-minimum structures of the three lipids in the
11PE series are illustrated in Fig. 6. One common
feature shared by them is the identical length of the ATS located in
the upper chain segment. As a result, the length of ATS alone cannot
account for the variations of Tm observed with
lipids in the
11PE 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 -CH2- units,
particularly those located far away from the chain methyl end, can make
contributions to the chain-melting process of trans
gauche isomerizations, resulting in a higher
Tm. 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 < Tm. 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
11PE series,
C(20):C(20:1
11)PE must, therefore, exhibit the highest
Tm. 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 C20-acyl chain of PE can
cause the bilayer to exhibit a higher Tm. As before,
this Tm elevating effect is small in comparison with
the opposing effect exerted by the reducing length of the chain segment
during acyl chain unsaturation. This is, however, not applicable in the
case of the C(20):C(20:2
11,14)PE
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
gauche isomerizations underlying the main phase transition at Tm. 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 -CH2- units
upon unsaturation at C(17) will not affect appreciably the Tm. As a result, the opposing effect of the rigid
triple
-bonds of C(20):C(20:3
11,14,17)PE becomes
dominant, and the relative magnitude of Tm between
C(20):C(20:2
11,14)PE and
C(20):C(20:3
11,14,17)PE thus becomes apparent. For the
three lipids in the
11PE series, their
Tm can thus be rationalized to have the following
decreasing order: C(20):C(20:1
11)PE > C(20):C(20:3
11,14,17)PE > C(20):C(20:2
11,14)PE. This order will yield a down and
up Tm curve in the plot of Tm
versus the number of
-bonds.
The
8PE series consists of the following four lipid
species: C(20):C(20:1
8)PE,
C(20):C(20:2
8,11)PE,
C(20):C(20:3
8,11,14)PE, and
C(20):C(20:4
8,11,14,17)PE. All of them share a common
upper chain segment of 5 consecutive methylene units extending from
C(3) to C(7) in the sn-2 acyl chain (Fig. 7). During the C(20):C(20:1
8)PE
C(20):C(20:2
8,11)PE conversion, the number of
-CH2- units in the ATS of the sn-2 acyl chain
decreases from 10 to 7, with the location of ATS being in the lower
chain segment. A reduction in Tm is thus expected to
accompany the conversion, and such a reduction has indeed been observed
calorimetrically. The subsequent C(20):C(20:2
8,11)PE
C(20):C(20:3
8,11,14)PE conversion is characterized by a
further shortening of three -CH2- units in the ATS of
cis,cis-8,11,-eicosadienoyl chain, leading to a continuous
decrease in Tm. In accompanying the
C(20):C(20:2
8,11)PE
C(20):C(20:3
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-8,11,14-eicosatrienoyl chain at C(17) affects only the length of the lower chain segment. In particular, the
length and position of ATS in the sn-2 acyl chain remain
unchanged as shown in Fig. 7, IV. Consequently, the trienoic
C(20):C(20:3
8,11,14)PE and the tetraenoic
C(20):C(20:4
8,11,14,17)PE share a common length of ATS
in their sn-2 acyl chains' upper segments. On the basis of
the identical length of ATS alone, the
Tm
associated with the C(20):C(20:3
8,11,14)PE
C(20):C(20:4
8,11,14,17)PE conversion would be zero. On
the other hand, the number of
-bonds in the sn-2 acyl
chain of C(20):C(20:4
8,11,14,17)PE is higher than that
in C(20):C(20:3
8,11,14)PE. As proposed earlier, this
additional
-bond may raise the Tm of
C(20):C(20:4
8,11,14,17)PE. Hence, on the basis of
the Tm elevating effect of multiple
-bonds and
the identical length of ATS, a positive
Tm can be
expected to underlie the C(20):C(20:3
8,11,14)PE
C(20):C(20:4
8,11,14,17)PE conversion. Indeed, the
expectation is borne out by DSC data presented in Fig. 3, in which the
Tm value displayed by the aqueous dispersion of
C(20):C(20:4
8,11,14,17)PE is seen to be 0.8 °C higher
than that of C(20):C(20:3
8,11,14)PE.
Three series of mixed-chain PEs with fixed numbers of
-bonds can be
seen along the diagonal lines in the Tm diagram (Fig. 3). The monoenoic PE series has a total number of five lipids. The di- and trienoic PE series consist of four and three lipids, respectively. In response to changes in the position of the double bond, the Tm values of lipids with a fixed number of double bonds give rise to a roughly V-shaped Tm
profile (Fig. 4, A and B). Molecular
interpretations of such a characteristic Tm profile
have been given in detail elsewhere from this laboratory (9). Hence, we
shall not discuss the V-shaped Tm profiles exhibited
by mono-, di-, and trienoic PE.
To sum up, for a series of sn-1 saturated/sn-2
unsaturated mixed-chain PE containing different numbers of
-bonds, a
continuously decreasing Tm profile is generally
observed in the plot of Tm versus the
number of
-bonds as exemplified by data shown in Figs. 1 or 2.
However, there are exceptions. For instance, the
C(20):C(20:2
11,14)PE
C(20):C(20:3
11,14,17)PE and the
C(20):C(20:3
8,11,14)PE
C(20):C(20:4
8,11,14,17)PE conversions are coupled with
increased Tm, and the Tm profiles
observed with lipids in the
8- and
11PE
series are thus characterized by a down and up trend. The mixed-chain PE that can, upon unsaturation, convert into a higher
Tm species has the following structural
characteristics: 1) the sn-2 acyl chain contains at least
two methylene-interrupted cis
-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
Tm values than their
6 precursors as shown
calorimetrically in the present and previous studies (8, 23). The
significance of the down and up Tm profile is that
it means the polyunsaturated
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.