(Received for publication, May 27, 1997)
From the Department of Biochemistry, Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908
In an attempt to examine the effects of different
numbers and positions of cis double bonds in the
sn-2-acyl chain of phosphatidylethanolamine (PE) on the
bilayer's melting behavior, 21 molecular species of PE were first
semisynthesized, and their Tm and H
values were subsequently determined by high resolution differential
scanning calorimetry. In the plot of Tm
versus the number of the cis double bond, some
characteristic profiles were observed for the various series of PEs.
For instance, if the cis double bond was first introduced
into the sn-2-acyl chain of C(20):C(20)PE at the
5-position, the Tm was observed to
reduce drastically. Subsequent stepwise additions of up to five
cis double bonds at the methylene-interrupted positions
toward the methyl end resulted in a progressive yet smaller decrease in
Tm. If, on the other hand, the cis
double bonds were introduced sequentially at the
11-,
11,14-, and
11,14,17-positions along the
sn-2-acyl chain of C(20):C(20)PE, the
Tm profile in the Tm
versus the number of the cis double bond showed
a down-and-up trend. Most interestingly, for positional isomers of
C(20):C(20:3
5,8,11)PE,
C(20):C(20:3
8,11,14)PE, and
C(20):C(20:3
11,14,17)PE, an inverted bell-shaped
Tm profile was detected in the plot of
Tm against the position of the
-carbon for these
isomers. Similar Tm profiles were also observed for
C(18):C(20)PE, C(20):C(18)PE, and their unsaturated derivatives. This
work thus demonstrated that both the positions and the numbers of
cis double bonds in the sn-2 acyl chain could
exert noticeable influence on the gel-to-liquid crystalline phase
transition behavior of the lipid bilayer. Finally, a molecular model
was presented, with which the behavior of the gel-to-liquid crystalline
phase transition observed for lipid bilayers composed of various
sn-1-saturated/sn-2-unsaturated lipids can be
rationalized.
In recent years, there has been increasing evidence suggesting the existence of a close correlation between some vital functions of cells and the various degrees of unsaturation in the sn-2-acyl chains of membrane phospholipids (1-3). Consequently, detailed and systematic investigation of the properties of sn-1-saturated/sn-2-unsaturated phospholipids in the organized membrane structure such as the lipid bilayer should be recognized and pursued. Furthermore, now is the time for studying unsaturated phospholipids, since sophisticated computer-based molecular modeling techniques from the hardware and the software fields have come together to provide valuable structural and energetic information about self-assembled biomolecules within a reasonable time frame. Results obtained with experimental and modeling studies of sn-1-saturated/sn-2-unsaturated phospholipids could eventually help illuminate the specific roles played by various natural phospholipids in many membrane-related cell functions or diseases.
The first detailed and systematic studies of the thermotropic phase behavior of phospholipids containing various numbers of cis double bonds in their sn-2-acyl chains were performed by Keough and co-workers (4-6). By using two series of phosphatidylcholines (PCs)1 and by applying the differential scanning calorimetry (DSC) techniques, they have shown that the introduction of a cis double bond into the sn-2-acyl chain at or near the center of the chain has a marked reducing effect on the phase transition temperature (Tm) associated with the gel-to-liquid crystalline phase transition of the lipid bilayer. A second cis double bond incorporated at the methylene-interrupted position toward the methyl end also reduces the Tm, but by a smaller amount. Interestingly, the introduction of a third cis double bond between the second cis double bond and the methyl end can cause a slight increase in Tm. This calorimetric work has been confirmed more or less by other investigators using PCs with different chain lengths (7, 8).
Recently, we have studied systematically the effect of different
positions of single cis double bond in the
sn-2-acyl chain on the phase transition behavior of lipid
bilayers composed of sn-1
saturated/sn-2-monounsaturated PCs or
phosphatidylethanolamines (PEs) by high resolution DSC and
computer-based molecular mechanics (MM) simulations (9-11). The
position of the single cis double bond (n) is
found in both lipid classes to exert a characteristic influence on the
gel-to-liquid crystalline phase transition temperature. Specifically,
in the plot of Tm versus
n, an
inverted bell-shaped profile is observed, and the minimum Tm occurs when the single cis double bond
is positioned at or near the center of the linear segment of the
sn-2-acyl chain. Furthermore, based on the results of MM
simulations, we have developed a molecular model that can explain the
characteristic Tm profile observed for monoenoic
phospholipids (10, 11).
In this study, we now extend our previous work by investigating the
gel-to-liquid crystalline phase transitions of one-component lipid
bilayers prepared from 21 molecular species of PE; these lipids contain
0-5 cis double bonds at different positions in the
sn-2-acyl chain. Specifically, we have examined the
influence of the positions of the cis double bonds, in
addition to the numbers of the cis double bond, on the phase
transition temperature of the lipid bilayer by high resolution DSC
techniques. In the plot of Tm versus the
number of the cis double bond, some characteristic profiles
are observed for the various series of PEs with different positions of
cis double bonds. Most interestingly, two parallel inverted
bell-shaped Tm profiles are detected in the plot of
Tm versus the position of the -carbon for two sets of positional isomers of
sn-1-saturated/sn-2-triunsaturated PEs. In
addition, we have codified and analyzed the published Tm values for lipid bilayers composed of various
sn-1-saturated/sn-2-unsaturated PCs. The
Tm dependence of the numbers and positions of the
cis double bonds in the sn-2-acyl chains of these
PCs shows a similar characteristic profile that parallels the behavior
of the corresponding PEs used in this study. This work thus
demonstrates that both the positions and the numbers of cis
double bonds in the sn-2-acyl chain can exert distinct
influence on the chain melting behavior of the PE and PC bilayers.
Moreover, we now expand the simple molecular model proposed earlier for
monoenoic lipids with different positions of the single cis
double bond in the sn-2-acyl chain. In particular, this
expanded molecular model takes the rigidity of multiple cis
double bonds into consideration, and it can be used to explain
adequately the characteristic chain melting behavior of lipid bilayers
containing various numbers and positions of cis double bonds
in the sn-2-acyl chains.
In this study, 21 molecular species of PE were semisynthesized. Three of them were saturated PE, and seven of them were either sn-1-saturated/sn-2-monounsaturated PE or sn-1-saturated/sn-2-diunsaturated PE. The semisynthesis of these 10 lipids were carried out using the established procedures as published previously (9, 12, 13). The other 11 molecular species were sn-1-saturated/sn-2-polyunsaturated PE containing three, four, and five cis double bonds at different positions in the sn-2-acyl chain. Initially, 11 molecular species of the corresponding sn-1-saturated/sn-2-polyunsaturated PC were semisynthesized and purified by established procedures (14), with a modified condition that all procedures were carried out as far as possible in an O2-free, N2 atmosphere to minimize autoxidation. This precautious step was also applied to the rest of the PE synthesis. The purified PCs (purity is greater than 98%) were then converted to PEs by transphosphatidylation with phospholipase D in the presence of excess amounts of ethanolamine hydrochloride, at pH 5.6, according to the procedure of Comfurius and Zwall (15) as previously published (12). The products were purified and separated by silica gel column chromatography, 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/sample was loaded on the thin layer plate. Possible oxidation of unsaturated fatty acyl chains was checked routinely as described earlier (13).
High Resolution DSC MeasurementDSC studies were performed
on a Microcal MC-2 microcalorimeter with a DA-2 digital interface and
data acquisition utility for automatic collection (Microcal, Inc.,
Northampton, MA), or a Hart 7708 differential scanning calorimeter
(Hart Scientific, Pleasant Grove, UT). In all experiments, a constant
heating scan rate of 15 °C/h was used, and 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 (9-11), the
gel-to-liquid crystalline phase transition temperature and transition
enthalpy were obtained from the second DSC heating curve. Specifically, the gel-to-liquid crystalline phase temperatures
(Tm) were taken from the transition peaks at the
maximum peak height positions, and transition enthalpies
(H) were calculated from the peak areas using software
provided by Microcal or Hart Inc.
We have
examined calorimetrically the gel-to-liquid crystalline phase
transition behavior of aqueous lipid dispersions prepared individually
from C(20):C(20)PE and its eight unsaturated derivatives. These nine
lipid species have a common structural feature; both the
sn-1- and sn-2-acyl chains contain 20 carbon
atoms. The structural difference among the nine lipid species lies in
the numbers and/or positions of the cis double bonds in
their sn-2-acyl chains. Fig. 1
illustrates the first and the immediate second DSC heating curves obtained with the aqueous lipid dispersions prepared individually from
these nine lipid species. It is evident that some lipid samples exhibit
thermal history-dependent phenomena. An example is
demonstrated by the C(20):C(20)PE sample. Initially, two
endothermic peaks centered at 66.3 and 82.7 °C are observed in
the first DSC heating scan as shown in Fig. 1. In general, the
formation of phospholipid bilayers in the crystalline state requires a
pronounced incubation time at temperatures considerably below
Tm. In our studies, phosphatidylethanolamines
constituting the bilayers in the aqueous dispersion are always
preincubated in the cold room (0 °C) for a minimum of 24 h.
Hence, the lipid bilayers in the aqueous dispersion have already
transformed into the highly ordered crystalline state prior to the
first DSC heating scan. As a result, the low and high temperature peaks
observed for C(20):C(20)PE in the first DSC heating scan correspond to
the crystalline-to-gel and gel-to-liquid crystalline phase transitions,
respectively (16). On immediate reheating, only the high temperature
peak at 82.5 °C is observed calorimetrically for the same
C(20):C(20)PE sample. Subsequent repeated reheatings result in, again,
a single endothermic transition at 82.5 °C, indicating that the
gel-to-liquid crystalline phase transition of the C(20):C(20)PE bilayer
is reproducible, and the crystalline-to-gel phase transition is
irreversible under the present experimental conditions. Another example
pertaining to the thermal history dependence of the phase transition
behavior is observed for the aqueous dispersion of
C(20):C(20:211,14)PE. In the first DSC heating scan, a
single, relatively sharp transition peaked at 26.4 °C is observed;
however, the same sample exhibits a broader transition centered at
22.4 °C upon immediate reheating (Fig. 1). A similar thermal
history-dependent phase transition behavior is also
exhibited by the sample of C(20):C(20:3
11,14,17)PE with
a single, sharp transition centered at 27.2 °C in the first DSC
heating scan and a broader transition peaked at 23.3 °C in the
second DSC heating scan (Fig. 1). In fact, such a thermal history-dependent behavior has been observed previously for
bilayers composed of saturated mixed chain PE (17) or saturated
identical chain PE containing short fatty acyl chains (18, 19). Based on the DSC results of these earlier studies, the single, sharp endotherm detected in the initial DSC heating scan of the
C(20):C(20:2
11,14)PE and
C(20):C(20:3
11,14,17)PE dispersions shown in Fig. 1 can
be ascribed to the crystalline-to-liquid crystalline phase transition,
and the broad low-temperature transition observed in the immediate
second and subsequent DSC heating scans shown in the same figure can be
assigned as the gel-to-liquid crystalline phase transition. Because of
the reproducible nature of the endothermic peak exhibited by the lipid
sample in the second and subsequent DSC heating scans, only the values
of the phase transition temperature (Tm) and the
transition enthalpy (
H) associated with the single
endotherms observed in the second DSC scans for various lipid samples
were measured; these experimentally determined values are summarized in
Table I.
|
The effects of numbers and positions of cis double bonds in
the sn-2-acyl chain of C(20):C(20)PE on the phase transition
temperature are illustrated in Fig. 2.
When the first cis double bond is introduced near the
carboxyl end of the sn-2-acyl chain at the 5
position, the Tm value is greatly reduced from 82.5 to 58.2 °C with respect to the Tm of the
saturated counterpart (Fig. 2A). When three cis
double bonds are present at the
5,8,11 positions, the
Tm is decreased by 35.2 °C from 58.2 to 23 °C.
As methylene-interrupted cis double bonds are further introduced sequentially into the sn-2-acyl chain of
C(20):C(20:3
5,8,11)PE on the methyl side of the newly
added cis double bond, the Tm value is
observed to decrease continuously, but by diminishing incremental
changes, from 23 to 6.6 and then to 3.5 °C (Fig. 2A). A
similar
H-lowering trend caused by each additional unsaturation is also observed for this series of lipids (Table I). It
should be noted that in this series of PE, the first cis double bond of all unsaturated sn-2-acyl chains begins at a
common position of
5 near the carboxyl end, and all
subsequent cis double bonds are introduced into the chain
segment located between the existing double bond and the methyl
terminus of the sn-2-acyl chain.
In the second series of C(20):C(20)PE derivatives presented in Fig.
2B, the first cis double bond is inserted at the
14 position in the sn-2-acyl chain. In
contrast to the unsaturated lipids shown in Fig. 2A, second
and subsequent cis double bonds are always introduced into
the chain segment located in between the existing double bond and the
carboxyl end. Hence, the sn-2-acyl chains of this series of
unsaturated phosphatidylethanolamines belong to the
-6 (or
n-6) family. The Tm value is shown in
Fig. 2B to decrease steadily with increasing numbers of
cis double bonds; however, the incremental change of the
Tm becomes progressively smaller. For comparison,
the Tm values of C(20):C(20)PC,
C(20):C(20:2
11,14)PC, and
C(20):C(20:4
5,8,11,14)PC reported by Keough et
al. (6) are also plotted against the number of cis
double bonds as indicated by the dotted curve in Fig.
2B. Most interestingly, the Tm-lowering
effect of each additional unsaturation appears remarkably similar to each other for the
-6 PE and
-6 PC series of lipids. In Fig. 2B, the Tm values for
C(20):C(20:1
14)PC and C(20):C(20:1
14)PE
bilayers, 26.9 and 46.1 °C, respectively, are calculated from equations derived previously for monoenoic PC and PE (20). Other Tm values of the various
-6 PEs are
experimentally obtained as listed in Table I. The enthalpy change
(
H) of the phase transition as a function of numbers of
cis double bonds follows a general trend similar to that
observed for Tm. The values of both
Tm and
H are summarized in Table
I.
A third series of PEs is presented in Fig. 2C. The first
cis double bond of all unsaturated PEs in this series of
lipids is positioned near the center of the sn-2-acyl chain
at the 11 position. Second and subsequent third
cis double bonds are added on the methyl side of the
existing double bond. As shown in Fig. 2C, the
Tm of the C(20):C(20:1
11)PE bilayer
is 39.2 °C lower than that of its saturated counterpart. A further
decrease of 20.9 °C in Tm is observed in going from the C(20):C(20:1
11)PE bilayer to the
C(20):C(20:2
11,14)PE bilayer. In contrast, a slight
increase in Tm (0.9 °C) is detected as the third
cis double bond is subsequently added into the dienoic
sn-2-acyl chain on the methyl end of the
14
double bond. The Tm values of C(20):C(20)PC and its various unsaturated derivatives have been determined calorimetrically by Keough et al. (6). In addition, the Tm
values of C(18):C(18)PC and its unsaturated derivatives with the first
cis double bond located near the center of the
sn-2-acyl chain at the
9 position have been
reported recently by Sanchez-Migallon et al. (7). These two
sets of calorimetric data obtained with PCs are connected by
dotted lines in Fig. 2C. Clearly, these two
dotted curves are strikingly similar in shape to the
solid curve determined in the present study using PEs. These
excellent agreements indicate that within this series of unsaturated
lipids the Tm value of the
-3-unsaturated lipid
with three cis double bonds is noticeably higher than that
of the
-6-unsaturated lipid with two cis double bonds,
regardless of the nature of the lipid's head group. Since the general
trends of the variations in Tm as a function of the
numbers of cis double bonds are different for the various
series of lipids shown in Fig. 2, A, B, and
C, it is thus concluded that the positions of cis
double bonds, in addition to the numbers of cis double
bonds, can influence the chain melting behavior of the lipid
bilayer.
To further substantiate the influence of the positions of
cis double bonds in the sn-2-acyl chains of PEs
on the gel-to-liquid crystalline phase transition behavior of the PE
bilayer, the Tm and H values of
bilayers composed of C(20):C(20:3
5,8,11)PE,
C(20):C(20:3
8,11,14)PE, and
C(20):C(20:3
11,14,17)PE are compared in Fig.
3. These three lipids species are
positional isomers; their numbers of carbon atoms and numbers of
cis double bonds are equal. The positions of the three
cis double bonds in the sn-2-acyl chain, however,
are different, and the difference can be represented by the position of
the first olefinic carbon counting from the methyl end (or the
-carbon). As shown in Fig. 3B, the Tm
values of these positional isomers have an inverted bell-shaped
dependence on the position of the
-carbon, with the minimal
Tm of 15.6 °C occurring at the
-6-position. To
the best of our knowledge, this is the first time that such an inverted
bell-shaped curve has been observed for the Tm values of lipid bilayers composed of positional isomers of
polyunsaturated phospholipids. Previously, it has been shown that the
Tm values of
sn-1-saturated/sn-2-monounsaturated PCs and PEs
with different positions of the single cis double bond
exhibit inverted bell-shaped profiles in the plot of
Tm versus the position of the single
cis double bond in the sn-2-acyl chain (9, 10). The Tm profile shown in Fig. 3B thus
demonstrates that, like monoenoic lipids, the melting behavior of
trienoic lipids is influenced in a distinctive manner by the position
of multiple cis double bonds along the sn-2-acyl
chain.
The Gel-to-Liquid Crystalline Phase Transition Behavior of Bilayers Composed of C(18):C(20)PE, C(20):C(18)PE, and Their Unsaturated Derivatives
In Fig. 1, the two acyl chains of various PEs all have 20 carbon atoms. To further investigate the influence of the introduction of increasing numbers of cis double bonds into the sn-2-acyl chain with a common position of the first cis double bond, we have utilized the high resolution DSC techniques to investigate the phase transition behavior of mixed chain C(18):C(20)PE, C(20):C(18)PE, and their unsaturated derivatives. In these mixed chain PEs, the two acyl chains are of unequal numbers of carbons; however, C(18):C(20)PE and C(20):C(18)PE are positional isomers in which the numbers of carbons in the sn-1 and sn-2-acyl chains of the first lipid species are equal to those in the sn-2- and sn-1-acyl chains, respectively, of the second lipid species.
Like the unsaturated derivatives of C(20):C(20)PE shown in Fig. 1,
lipid bilayers prepared from some of the unsaturated derivatives of
mixed chain PE also exhibit different phase transition behavior in the
first and the second DSC heating scans (data not shown). For instance,
the C(18):C(20:211,14)PE bilayer exhibits a single,
narrower endotherm peaked at 24.2 °C in the first DSC heating scan,
and this we ascribe to the crystalline-to-liquid crystalline phase
transition. Upon reheating, this taller endotherm is replaced by a
broader transition centered at 18.5 °C, and this is assigned as the
gel-to-liquid crystalline phase transition. Similarly, we find that the
single transition peak corresponding to the crystalline-to-liquid
crystalline phase transition of the C(20):C(18:2
9,12)PE
bilayer is narrower, with a Tm of 12.4 °C in the initial DSC heating scan. Upon immediate reheating, the same bilayer exhibits a broader gel-to-liquid crystalline phase transition with
Tm of 7.2 °C. The bilayer composed of trienoic
-3 lipids such as C(18):C(20:3
11,14,17)PE or
C(20):C(18:3
9,12,15)PE also shows a similar thermal
history-dependent behavior. Nevertheless, the single
transition that appears in the second DSC heating scan is always
reproducible upon repeated reheatings, and we assign it as the
gel-to-liquid crystalline phase transition. In Table I, the
Tm and
H values of C(18):C(20)PE,
C(20):C(18)PE, and their unsaturated derivatives are obtained from the
gel-to-liquid crystalline phase transitions that appeared in the second
DSC heating scans.
Fig. 4A depicts the plot of
Tm versus the increasing numbers of
cis double bonds in the sn-2-acyl chain of
C(18):C(20)PE. In this series of unsaturated PE, the common
cis double bond in the sn-2-acyl chains is
located at the 14 position, and the additional double
bonds are on the carboxyl side of the existing double bond; hence, this
series of unsaturated lipids belongs to the
-6 family. The data
shown in Fig. 4A demonstrate clearly the
Tm-lowering effect of each additional unsaturation. This decreasing trend in Tm is virtually identical
to that of the solid Tm curve shown in Fig.
2B for the unsaturated derivatives of C(20):C(20)PE. We
should point out here that the Tm value of the
C(18):C(20:1
14)PE bilayer, 44.5 °C, is a calculated
one (20). The literature values of Tm for bilayers
composed of C(18):C(20)PC, C(18):C(20:1
14)PC,
C(18):C(20:2
11,14)PC,
C(18):C(20:3
8,11,14)PC, and
C(18):C(20:4
5,8,11,14)PC are 60.4, 25.1,
5.4,
9.3,
and
13.2 °C, respectively (8, 17, 20). These Tm
values are connected by a dotted line in the plot of
Tm versus the number of double bonds as
shown in Fig. 4A. Interestingly, this dotted
curve appears remarkably similar in shape to the solid
curve obtained with C(18):C(20)PE and its unsaturated derivative.
It should be noted that, as described earlier, the solid and
dotted curves connecting Tm values of PEs
and PCs, respectively, in Fig. 2, B and C, are
also roughly parallel, suggesting strongly that the
Tm-lowering effects of chain unsaturations in both
PE and PC bilayers perhaps proceed by a common mechanism.
In Fig. 4B, the sn-2-acyl chains of the
unsaturated derivatives of C(18):C(20)PE have a common cis
double bond at the 11 position; in addition, the second
and subsequent methylene-interrupted cis double bonds are
inserted between the existing double bond and the methyl end of the
sn-2-acyl chain. In the plot of the Tm
versus the number of cis double bonds, the
Tm value decreases initially with increasing numbers
of cis double bonds. Beyond two cis double bonds,
the magnitude of Tm increases slightly.
Specifically, the Tm of the trienoic
-3 lipid,
C(18):C(20:3
11,14,17)PE, is 21.0 °C, which is
2.5 °C higher than that of the dienoic
-6 lipid,
C(18):C(20:2
11,14)PE. As expected, the same down-and-up
trend in Tm is also observed in Fig. 4B
for the mixed chain C(16):C(18)PC series, in which the first
cis double bond in the shorter sn-2-acyl chain is
inserted at the
9 position and the subsequent additions
of cis double bonds are on the methyl side of the existing
double bond. The Tm values of C(16):C(18)PC and its
unsaturated derivatives that appeared in Fig. 4B were those
reported by Hernandez-Borrell and Keough (21) and McCabe et
al. (22).
The H and Tm values of lipid bilayers
prepared from C(18):C(20:3
5,8,11)PE,
C(18):C(20:3
8,11,14)PE, and
C(18):C(20:3
11,14,17)PE are plotted in Fig. 3,
A and B, against the position of the
-carbon
of the three positional isomers. An inverted bell-shaped Tm profile is observed in Fig. 3B with
the minimal Tm of 11.7 °C occurring at the
-6
position. The structural difference among these three isomers lies only
in the position of the methylene-interrupted multiple cis
double bonds in the sn-2-acyl chain. The observed Tm profile thus provides further strong evidence
indicating that the positions of cis double bonds alone can
exert a noticeable influence on the lipid bilayer's transition
behavior.
Fig. 5, A and B,
show the variations of Tm as a function of the
number of cis double bonds in the sn-2-acyl
chains of C(20):C(18)PE and its unsaturated derivatives. In Fig.
5A, the continuous downshift in Tm
arising from a series of -3 PEs with increasing numbers of
cis double bonds is, in fact, another recurring theme that
has been observed repeatedly for
-6 PEs and PCs as depicted in Figs.
2B and 4A. Here, 46.9 °C is calculated as the
Tm value for the C(20):C(18:1
15)PE
bilayer (20). The Tm curve illustrated in Fig. 5B, on the other hand, shows a somewhat more complicated,
yet familiar, picture; the Tm of C(20):C(18)PE
decreases steadily with increasing numbers of cis double
bonds, reaching a nadir at two cis double bonds. Thereafter,
the incremental difference in Tm changes its sign,
resulting in a Tm curve with a characteristic
down-and-up trend. It should be noted that the same down-and-up trend
in Tm has already been shown to associate with the
gel-to-liquid crystalline phase transition behavior of some other
series of PE and PC as illustrated in Figs. 2C and
4B. Let us focus now upon the common sequence of events shared by these different series of phospholipids, leading to the
characteristic down-and-up trend in Tm. First, the introduction of the initial cis double bond into the
saturated sn-2-acyl chain of PE or PC occurs at or near the
center of the acyl chain. Second, the subsequent incorporation of
methylene-interrupted cis double bonds takes place between
the existing double bond and the methyl terminus of the
sn-2-acyl chain. Third, the lipid species showing an upward
trend with a positive incremental change in Tm is
inevitably an
-3 lipid.
When the lipid bilayer prepared from a one-component phospholipid
species undergoes the thermally induced gel-to-liquid crystalline phase
transition, an equilibrium state is reached between the gel phase and
the liquid crystalline phase at the phase transition temperature,
Tm. Assuming the transition to be a first-order equilibrium transition, the Tm is then equal to the
ratio of the transition enthalpy and the transition entropy as follows: Tm = H/
S. Based on the
experimentally determined values of Tm and
H, the value of
S can be calculated
accordingly. In Table I, the values of Tm,
H, and
S for the one-component lipid
bilayers prepared individually from several series of PEs are
summarized. It is evident that within each series of PEs the magnitude
of Tm,
H, and
S are
clearly dependent on the numbers and positions of cis double
bonds in the sn-2-acyl chains. Moreover, when the
Tm value is diminished as a result of chemical
modification of the sn-2-acyl chain, the values of the
accompanying
H and
S are, in general, also
observed to decrease. This means a greater contribution of the energy
term
H to the magnitude of Tm, since
the other term,
S, works against
H in the
relationship of Tm =
H/
S. Consequently, as a first
approximation, the term Tm may be expressed explicitly as a function of some structural parameters that are directly related to the energetic term
H, without
invoking the
S term. Specifically, the gel-to-liquid
crystalline phase transition is accompanied structurally by a highly
cooperative process involving the trans
gauche isomerizations of the C-C single bonds along the two
acyl chains within each lipid molecule in the bilayer. The
calorimetrically determined
H, on the other hand,
represents a measure of heat required to overcome the energy barrier
for the gel-to-liquid crystalline phase transition. This heat must therefore depend on the total number of C-C trans bonds,
which in turn is related directly to the effective chain lengths of the
lipid's acyl chains in the gel state bilayer. As a result, the
variation in Tm for lipid bilayers prepared
individually from a series of positional isomers for a given lipid
species may be correlated with the change in the effective acyl chain lengths of these positional isomers. Based on this possible correlation and the general molecular structure of monoenoic lipid obtained from MM
calculations, a simple molecular model has been developed to describe
the gel-to-liquid crystalline phase behavior of bilayers prepared from
a series of monounsaturated lipids with different positions of the
single cis double bond in the sn-2-acyl chain (9-11); specifically, three basic assumptions underlying this simple
model have been proposed, and they are briefly summarized in the next
paragraph. Subsequently, an expanded molecular model is further
proposed. The phase transition behavior of lipid bilayers composed of
multiple cis double bonds as presented under "Results" can then be rationalized.
The three basic assumptions underlying the simple molecular model are
as follows: 1) the monoenoic sn-2-acyl chain in the sn-1-saturated/sn-2-monounsaturated phospholipid
molecule is assumed to adopt, at T < Tm, an energy-minimized crankshaft-like motif in the
gel state bilayer; hence, it consists of a longer chain segment and a
shorter chain segment separated by the cis double bond; 2)
the longer segment and the neighboring all-trans sn-1-acyl
chain run in a parallel manner with favorable van der Waals attractive
distance between them; and 3) the shorter segment is considered to be
partially disordered at T < Tm,
analogous to the molten polypeptide chain of proteins, thus playing a
relatively insignificant role in the attractive van der Waals
chain-chain interactions in the gel state bilayer. With this model, it
can be appreciated that the total number of C-C trans bonds
in the two acyl chains of monounsaturated lipids in the gel state
bilayer is considerably smaller than that of the saturated counterparts due to the third assumption that the short segment is already partially
disordered at T < Tm. As a result,
the heat required to induce the cooperative process of trans
gauche isomerizations of C-C single bonds in the two
acyl chains of self-assembled monounsaturated lipid molecules is
decreased appreciably, resulting in a significantly lower
Tm value. In addition, we can use the same molecular model to explain the inverted bell-shaped Tm profile observed for bilayers of monoenoic phospholipids in the plot of Tm versus the position of the single
cis double bond in the sn-2-acyl chain. As stated
in the second assumption, the longer chain segment of the kinked
sn-2-acyl chain is proposed to undergo a favorable van der
Waals contact interaction with the all-trans sn-1-acyl chain
in the gel state bilayer. This contact interaction energy must then
depend on the effective chain length of the longer segment. When the
single cis double bond is positioned at the center of a
sn-2-acyl chain, the effective chain length of the longer
segment has a minimum length, which is almost equal to that of the
shorter segment. Hence, the van der Waals contact interaction with the
all-trans sn-1-acyl chain in the gel state bilayer is also
minimal. As the single cis double bond migrates away
successively from the chain center toward either the carboxyl or the
methyl end, the effective length of the longer segment is progressively
increased, leading to a proportionally increased van der Waals
interaction and hence a gradual increase in Tm and
H. The inverted bell-shaped profile of
Tm as a function of the location of the
cis double bond can thus be envisioned based on the simple
structural model. Specifically, we can take a saturated
sn-2-acyl chain containing 20 carbons in C(20):C(20)PC as an
example. Here, the effective chain length of this sn-2-acyl chain in the gel state bilayer of C(20):C(20)PC is about 17.5 C-C bond
lengths. This effective length is approximately 1.5 C-C bond lengths
shorter than that of the fully extended chain, resulting from the
initial sharp bend of the sn-2-acyl chain at the C(2) position. Now, if a single cis double bond is inserted at
the
11 position, the kinked monoenoic acyl chain is then
shortened by about one C-C unit (11). The effective length of the
longer segment preceding the cis double bond at the
11 position is 8.5 C-C bond lengths, and that of the
shorter segment succeeding the cis double bond is 8 C-C
bond lengths (Fig. 6). If the single
cis double bond migrates from the
11 position
toward the carboxyl end and stops at the
5 position, the
effective chain lengths of the longer and shorter segments are changed
to 14 and 2.5 C-C lengths, respectively (Fig. 6). On the other hand,
if the single cis double bond migrates to the
14 position, the longer and shorter segments will have
11.5 and 5 C-C bond lengths, respectively (Fig. 6). Clearly, if a
simple correlation between the Tm and the length of
the longer chain segment of the sn-2-acyl chain is assumed
to exist for this series of positional isomers, the
Tm value of C(20):C(20:1
n)PC isomers
should have the following increasing order:
11 <
14 <
5. Indeed, this trend of
Tm is borne out by experimental and computational
data (20). This means that our proposed simple correlation between the
Tm and the effective chain length of the longer
segment of the monoenoic sn-2-acyl chain for this series of
positional isomers is reasonable.
Now we can expand the molecular model discussed above and then apply the expanded model to lipids containing more than one single cis double bond. First of all, it should be mentioned that when a second cis double bond is introduced into the monoenoic sn-2-acyl chain at the methylene-interrupted position, the dienoic chain can still adopt the crankshaft-like motif as indicated by MM calculations (13). In addition, the effective length of the kinked sn-2-chain is reduced by another C-C unit, and the axes of the two chain segments separated by the two cis double bonds are roughly parallel (13). Furthermore, the vertical distance separating the two parallel axes of the chain segments is larger than that observed for monoenoic chain (13, 23), leading to a weaker lateral chain-chain interaction between the sn-1- and sn-2-acyl chain in the gel state bilayer. In comparison with other sn-1-saturated/sn-2-polyunsaturated lipids, we propose that in the gel state bilayer the overall lateral chain-chain interaction is minimal for bilayers composed of sn-1-saturated/sn-2-diunsaturated lipids. This proposal is based on a poignantly paradoxical nature of the cis double bond: the cis double bond itself is rotationally immobile, whereas the two C-C single bonds adjacent to the cis double bond are highly flexible (23). More specifically, the rigidity and the flexibility of the sn-2-acyl chain at and around the cis double bond can work against each other in terms of lateral chain-chain interactions. When there are two cis double bonds, the dienoic sn-2-acyl chain can be considered to be highly dynamical as a result of the flexible nature of the C-C single bonds adjacent to the two cis double bonds, thus resulting in a weaker lateral chain-chain interaction between the sn-1- and sn-2-acyl chains. After the second cis double bond, however, the rigidity of the multiple cis bonds is considered to play a more dominant role, thus promoting the lateral chain-chain interaction. In fact, for sn-1-saturated/sn-2-polyunsaturated lipids packed in the gel state bilayer, the multiple methylene-interrupted cis double bonds are assumed to form a structural unit with highly restricted mobility. Consequently, for a series of positional isomers of lipids containing the same number of multiple cis double bonds, the change in Tm as a function of the position of the immobile unit in the sn-2-acyl chain can be expected to correlate closely with the corresponding change of the chain length of the longer segment of the kinked sn-2-acyl chain. The Tm profile should, therefore, have an inverted bell-shaped characteristic, similar to that observed for monoenoic lipids with different positions of the single rigid cis double bond. And indeed, this characteristic profile is observed experimentally as shown in Fig. 3B.
We reemphasize that our expanded molecular model is constructed on the
basis of the following two additional assumptions: 1) the
sn-2-acyl chain containing two cis double bonds
is highly flexible in the gel state bilayer, leading to a weakest
lateral chain-chain interaction in comparison with other
sn-1-saturated/sn-2-polyunsaturated lipids; 2)
when the sn-2-acyl chain contains three or more
cis double bonds, however, these methylene-interrupted
cis double bonds can be considered as an essentially
immobile unit in the gel state bilayer. Based on the expanded molecular
model, the variations in Tm for various unsaturated
lipids with different positions of the cis double bonds can
be readily correlated with the effective chain lengths of the longer
segments of the sn-2-acyl chains. For instance, the
effective chain lengths of the longer segments of the
sn-2-acyl chains for C(20):C(20:15)PE,
C(20):C(20:3
5,8,11)PE,
C(20):C(20:4
5,8,11,14)PE, and
C(20):C(20:5
5,8,11,14,17)PE are shown in Fig. 6 to be
14, 8, 5, and 2.5 C-C bond lengths, respectively. For this series of
lipids, the Tm can then be expected to decrease
continuously with an increasing number of cis double bonds,
and the expectation is indeed borne out by experimental data (Fig.
2A). The effective chain lengths of the longer segments of
the sn-2-acyl chains for the various
-6 lipids of
C(20):C(20:1
14)PE, C(20):C(20:2
11,14)PE,
C(20):C(20:3
8,11,14)PE, and
C(20):C(20:4
5,8,11,14)PE in the gel state bilayer are
11.5, 8.5, 5.5, and 5 C-C bond lengths, respectively (Fig. 6). As
expected, the Tm also shows a trend that roughly
parallels the chain length variation of the longer segment of the
kinked chain (Fig. 2B). In Fig. 2C, a down-and-up
trend of the Tm profile is observed for C(20):C(20:1
11)PE, C(20):C(20:2
11,14)PE,
and C(20):C(20:3
11,14,17)PE. The lengths of the longer
segments of the sn-2-acyl chains of this series of lipids
are identical, being 8.5 C-C bond-lengths (Fig. 6). However, the
rigidity of the sn-2-acyl chain containing three
cis double bonds is greater than that of the dienoic
sn-2-acyl chain, leading to a somewhat stronger lateral
chain-chain interaction between the sn-1 and
sn-2-acyl chains and hence a slightly higher Tm. Likewise, the general trend of the experimental
Tm values of C(18):C(20)PE derivatives observed in
Fig. 4A also follows, in a parallel manner, the variation in
the apparent chain length of the longer segment of the
sn-2-acyl chain among the various lipids (Fig. 6). The
down-and-up trend of Tm observed in Fig.
4B, however, can be explained by the weakest chain-chain interaction proposed for dienoic lipids in the gel state bilayer.
For the various C(20):C(18)PE derivatives containing 18 carbons and
various numbers and positions of cis double bonds in the sn-2-acyl chain, the effective lengths of the longer and
shorter segments of the sn-2-acyl chains in the gel state
bilayer are illustrated in Fig. 7. The
experimental curves in the two plots of Tm
versus the numbers of cis double bonds shown in Fig. 5, A and B, can also be explained
satisfactorily based on the variations in the chain length of the
longer segment of the sn-2-acyl chain among the various
lipids as depicted in Fig. 7 and the increased rigidity of the
sn-2-acyl chain resulting from an increased number of
cis double bonds. Finally, it should be emphasized that the
simple correlation between the Tm and the chain
length of the longer segment of the sn-2-acyl chain in the
gel state bilayer proposed in this study for various series of
sn-1-saturated/sn-2-unsaturated PE (or PC) should
be used with caution. It can be applied only to describe qualitatively
the trend of Tm variations as shown in Figs. 2, 3, 4, 5. It should not be applied to estimated the magnitude of
Tm in any of the Tm profiles.
Phosphatidylethanolamines with a given pair of
sn-1-saturated/sn-2-unsaturated acyl chains
packed in the lipid bilayer can undergo a gel-to-liquid crystalline
phase transition upon heating. The thermodynamic behavior of this phase
transition is different from that displayed by saturated counterparts.
In general, the values of Tm, H, and
S associated with the gel-to-liquid crystalline
transition are noticeably smaller for the bilayer composed of
unsaturated lipid species.
The change in Tm upon the introduction of additional cis double bonds into the sn-2-acyl chain depends on both the numbers and the positions of cis double bonds along the sn-2-acyl chain.
In the presence of cis double bonds, the sn-2-acyl chain of sn-1-saturated/sn-2-unsaturated PE (or PC) is proposed to have a crankshaft-like motif at T < Tm. With this motif, the sn-2-acyl chain appears to consist of two chain segments with nearly parallel axes separated by the cis double bond(s). Furthermore, the longer segment and the neighboring all-trans sn-1-acyl chain are assumed to undergo favorable van der Waals interactions, whereas the shorter segment is assumed to be partially disordered at T < Tm.
Based on the structural model proposed above, the trend of Tm variations for a series of sn-1-saturated/sn-2-unsaturated PE (or PC) in the plot of Tm against the numbers of cis double bonds can be correlated directly with the change in the effective length of the longer segment of the kinked chain. If the lengths of the longer segments are identical for a series of lipids, an increase in the rigidity of the polyunsaturated sn-2-acyl chain should be taken into consideration. In this case, the dienoic lipid has a minimal Tm value; thereafter, the Tm increases slightly with increasing numbers of cis double bonds.
The worth of a molecular model lies in its predictive power. With our molecular model, we predict that in the plot of Tm versus the position of two cis double bonds for a series of positional isomers of sn-1-saturated/sn-2-diunsaturated PE (or PC), the Tm profile should exhibit an inverted bell-shaped characteristic. Only future experiments with dienoic lipid species can test the predictive ability of our proposed structural model.
This paper is dedicated to Professor Thomas E. Thompson on the occasion of his retirement.