(Received for publication, June 5, 1995; and in revised form, July 21, 1995)
From the
Recently, we have shown by high resolution differential scanning
calorimetry that the position of a cis double bond
(-bond) in a series of 1-stearoyl-2-octadecenoyl-
phosphatidylcholines can affect the phase transition temperature (T
) or enthalpy (
H) of the
gel-to-liquid crystalline phase transition of this series of lipids in
the following manner. The value of T
(or
H) is minimal when the
-bond is positioned at C(11)
in the sn-2 acyl chain; in addition, this value increases
steadily as the
-bond migrates toward either end of the acyl
chain, resulting in a symmetrical, inverted bell-shaped profile (Wang,
Z.-q., Lin, H.-n., Li, S., and Huang, C. (1995) J. Biol. Chem. 270, 2014-2023). In this communication, we have further
demonstrated the inverted bell-shaped profile of T
using 1-arachidoyl-2-eicosenoyl-phosphatidylcholines. In
addition, we have extended the lipid series of
1-stearoyl-2-octadecenoyl-phosphatidylcholines to include
1-arachidoyl-2-octadecenoyl- phosphatidylcholines and
1-behenoyl-2-octadecenoyl-phosphatidylcholine, each series with a
-bond at varying carbon position of 6, 7, 9, 11, 12, and 13.
Calorimetric results obtained with these three series of lipids
indicate that the inverted bell-shaped curve shifts toward higher
temperatures in a nonuniform manner as the saturated sn-1 acyl
chain length increases from 17 to 19 and then to 21 C-C bond
lengths. Specifically, the T
(or
H) values are nearly identical for these cis-monoenoic lipids when their
-bonds are positioned at
C(13). Based on the height of the rotational energy barrier obtained
with molecular mechanics calculations, it is evident that the
rotational flexibility of the single C-C bond adjacent to the
-bond in 1-stearoyl-2-octadecenoyl-phosphatidylcholine increases
as the
-bond migrates from C(9) to C(13). The differential
scanning calorimetry results obtained with the three series of lipids
can thus be attributed to an increase in the rotational flexibility of
the short chain segment succeeding the C(14) atom in the sn-2
octadecenoyl chain. In this communication, we also propose that in the
gel-state bilayer of sn-1 saturated/sn-2 cis-monounsaturated phosphatidylcholine the entire length of
the shorter segment of the sn-2 acyl chain acts as a
structural perturbing element; hence, it is mainly responsible for the
large lower T
of the monoenoic lipid
relative to the saturated counterpart. Finally, two general equations
relating T
with the structural parameters
of cis-monoenoic phosphatidylcholines are presented. These
equations, formulated primarily on the assumption that the short
segment of the sn-2 acyl chain acts as a perturbing element,
are shown to have strong predictive power in estimating the T
values of the gel-to-liquid crystalline
phase transitions for sn-1 saturated/sn-2 cis-monounsaturated phosphatidylcholines.
Phosphatidylcholines isolated from the plasma membrane of eukaryotic cells are a structurally diverse group of phospholipids. The bewildering variety of membrane phosphatidylcholines originates from the numerous possible combinations of sn-1 and sn-2 acyl chains, most of which are derived biosynthetically from saturated and unsaturated fatty acyl-CoA, respectively. By and large, the acyl chain lengths and chemical structures of the two acyl chains in a membrane phosphatidylcholine molecule are different. A good example is 1-palmitoyl-2-arachidonoyl-phosphatidylcholine, one of the most abundant lipid species found in liver cells. In the plasma membrane, phosphatidylcholine molecules aggregate in the form of the lipid bilayer due to their amphipathic nature, thus constituting the basic structural matrix. In addition, some phosphatidylcholine molecules serve as the metabolic precursors of intrinsic signaling elements, thus conferring some regulatory properties on eukaryotic cells. Consequently, it is important and relevant to investigate the intricate relationships between the structure and properties of the lipid bilayer composed of naturally occurring phosphatidylcholines.
Although the structures of a large number of phospholipids with saturated and identical number of carbon atoms in their two acyl chains have been determined by crystallographic approaches(1) , the prevalence of the single-crystal structures of naturally occurring phospholipids is still elusive. However, computer-based molecular modelings for biomolecules have been advanced rapidly in recent years. This approach offers the possibility of simulating the unknown structure of naturally occurring phospholipids based on the single-crystal structures of saturated phospholipids(2) . The combination of this computational approach together with calorimetric data, for example, has provided valuable information relating the structure and melting behavior of naturally occurring phospholipids in the bilayer(3, 4) .
In this communication, the
thermotropic phase behavior of 26 molecular species of
phosphatidylcholine with sn-1 saturated/sn-2 cis-monounsaturated acyl chains was studied by high resolution
differential scanning calorimetry (DSC). ()These lipids were
semisynthesized in this laboratory; however, they all resemble strictly
the naturally occurring monoenoic phosphatidylcholines. The structures
of these cis-monoenoic phosphatidylcholines were simulated
using the molecular mechanics method, specifically the MM3(92) force
field(5) . It is well known that fully hydrated cis-monoenoic phosphatidylcholines can exhibit
calorimetrically a characteristic T
of
the gel-to-liquid crystalline phase transition; moreover, this T
is always far below that of the
saturated counterpart(6, 7) . We have undertaken in
this work the combined approach of DSC and MM methods in order to gain
a deeper understanding of the difference in T
between the cis-monoenoic phosphatidylcholine and
the saturated counterpart. Also, we will show in this work that
quantitative equations relating T
with
the structural parameters for cis-monoenoic
phosphatidylcholines can be developed; these equations allow us to
predict the T
values for cis-monoenoic phosphatidylcholines in general.
In modeling the various structures of cis-monoenoic phosphatidylcholine molecules, the structure of the diacyl moiety of 1-palmitoyl-2-oleoyl-phosphatidylcholine with a type IIIb kink motif (2) was used as the starting point; in addition, the headgroup structure of the monoenoic phosphatidylcholine molecule was assumed initially to be identical to that of the dihydrate of dimyristoyl phosphatidylcholine, Structure B, obtained by x-ray diffraction(10) . For instance, the initial crude structural model of 1-arachidoyl-2-gondoyl-phosphatidylcholine was constructed as follows: four methylene units, each being linked by the trans C-C bond, were first added to the sn-1 acyl chain of 1-palmitoyl-2-oleoyl-phosphatidylcholine with a type IIIb kink motif. Then, two methylene units were added to the upper segment of the sn-2 acyl chain to form the C(3)-C(4) segment. The resulting crude structure of 1-arachidoyl-2-gondoyl-phosphatidylcholine was subsequently refined by subjecting to energy minimization using Allinger's MM3 program. In order to ensure that the minimum in the potential energy surface was practically reached, additional rounds of energy minimizations were routinely performed. The application of Allinger's program in determining the structure of lipids and the rotational energy barrier of the C-C bond in lipid acyl chain was discussed in detail elsewhere(2) . Here, it is worth pointing out the reason why 1-palmitoyl-2-oleoyl phosphatidylcholine with a type IIIb kink was chosen as the starting point for the construction of other monoenoic lipid molecules. This is due to the excellent agreement between the computational structure obtained by MM approach and the reconstructed structure based on x-ray diffraction data detected for this particular conformation of 1-palmitoyl-2-oleoyl-phosphatidylcholine(2) .
Before we present our experimental and computational results,
it is appropriate to mention first the rules of nomenclature for
abbreviating diacyl phospholipids adopted in this study. For a
saturated diacyl lipid species, C(X):C(Y)PC denotes a
phosphatidylcholine (PC) molecule with X and Y carbon
atoms in the sn- 1 and sn-2 acyl chains,
respectively; hence, the notation C(X) preceding the colon in
C(X):C(Y)PC refers to the sn-1 acyl chain
with X carbons, and the notation C(Y) succeeding the
colon gives the total number of carbons (Y) in the sn-2 acyl chain. The convention for numbering the carbon atom
in the acyl chain begins at the carboxyl end. The sn-2 acyl
chain of a saturated diacyl C(20):C(18)PC, for instance, has 18 carbon
atoms; its carbonyl carbon is C(1) and its terminal methyl carbon is
C(18). For a sn-1 saturated/sn-2 monounsaturated
phosphatidylcholine molecule, it is abbreviated as
C(X):C(Y:1)PC. Here, we designate
the position of the cis double bond as
,
where the superscript n refers to the lower number of the two
carbon atoms linked by the double bond. For instance, the double bond
at the C(9)=C(10) position in the oleoyl chain is designated by
. The numerical value 1 after the colon in the
notation C(Y:1
) refers to a single cis carbon-carbon double bond (
) at the n position along
the sn-2 acyl chain. For
1-arachidoyl-2-gondoyl-phosphatidylcholine and
1-palmitoyl-2-oleoyl-phosphatidylcholine, they can thus be abbreviated
as C(20):C(20:1
)PC and
C(16):C(18:1
)PC, respectively.
Figure 1:
Thermal behavior exhibited
by a series of cis-monoenoic phosphatidylcholines with
different -bond positions. A, representative DSC heating
thermograms for aqueous dispersions of
C(20):C(20:1
)PC with n = 5,
8, 11, and 13. The thermograms were the second DSC heating scans
obtained at a constant scan rate of 15 °C/h. Lipid concentration:
3-6 mM. Each aqueous lipid dispersion contains 5 mM phosphate buffer (pH 7.4), 1 mM EDTA, and 50 mM
NaCl. B, the plot of T
versus the
-bond position,
, for the series of monoenoic lipids depicted
at the left.
Fig. 1B shows the T value as a
function of the
-bond position,
, for the series
of lipids shown in Fig. 1A. It is evident that a steady
migration of the
-bond from C(5) to C(8) and then to C(11) results
in a progressive decrease in T
. However, the
incremental drop in T
does not correspond
proportionally to the stepwise increase in the
position.
Interestingly, as the
-bond migrates further down along the sn-2 acyl chain from C(11) to C(13), the T
value increases slightly. The smooth parabolic curve
connecting the four data points seen in Fig. 1B is the
least-squares fitting curve. This parabolic character of the T
versus
-position curve
is in close agreement with previous DSC studies using
C(18):C(18:1
)PC with n = 6, 7, 9, 11,
12, and 13(4) . The transition enthalpy (
H)
associated with the chain melting transition is calculated from the
area under the endothermic peak, and the
H values for
aqueous dispersions prepared from the four isomers of
C(20):C(20:1
)PC under study are summarized in Table 1. The change in
H as a function of the
position is observed to follow, within experimental
errors, the same general trend as that of the T
.
However, due to the large scattering of the experimental data, a clear
minimum in
H at n = 11 in the
Hversus
position curve is not
discernible (Table 1).
Of the four heating thermograms shown
in Fig. 1A, the one exhibited by the aqueous dispersion
of C(20):C(20:1)PC shows a single transition with the
sharpest endothermic peak centered at 30.7 °C and a peak width at
half-height (
T) of 0.3 °C. On cooling, however, the
exothermic transition of the same lipid sample gives rise to a peak
centered at 30.7 °C with a discernible shoulder at 31.1 °C (DSC
curve not shown). The molecular origin of the shoulder is uncertain;
nevertheless, it seems that an obligatory and transient intermediate
state may exist at 31.1 °C for the C(20):C(20:1
)PC
bilayer as the bilayer converts, upon cooling, from the
liquid-crystalline state to the gel state. One may further speculate
that monoenoic lipids in the putative state of the transient
intermediate are likely to have such characteristic features that the
upper and lower segments of the sn-2 acyl chain separated by
the
-bond are in the order (or gel) and disorder (or
liquid-crystalline) states, respectively.
Fig. 2shows the representative DSC heating thermograms
for C(20):C(18:1)PC and
C(22):C(18:1
)PC with n = 6, 7, 9, 11,
12, and 13. Most thermograms are characterized by a highly cooperative
endothermic transition, which can be readily assigned as the chain
melting or the gel-to-liquid crystalline phase transition. Of those
with two endotherms, the larger transition always occurs at a higher
temperature, which is assigned as the gel-to-liquid crystalline phase
transition. This assignment is supported by the observation that
transition characteristics of this high temperature endotherm are
reproducible after repeated cooling/heating cycles, whereas those of
the low temperature transition are thermal-history dependent. For
example, the C(20):C(18:1
)PC dispersion exhibits two
overlapped transitions (Fig. 2). The high temperature transition
peaked at 8.5 °C and is reproducible upon repeated heatings or
coolings. The low temperature transition, however, has a peak at 7.2
°C upon heating; this peak is shifted to 7.0 °C upon cooling.
We, therefore, assign the high temperature transition as the
gel-to-liquid crystalline phase transition for fully hydrated
C(20):C(18:1
)PC with a T
of 8.5
°C. The
H value of C(20):C(18:1
)PC
is calculated based on the overlapped peak area of the two transitions
shown in the DSC heating curve. The values of thermodynamic parameters (T
,
H,
S, and
T)
associated with the gel-to-liquid crystalline phase transition for all cis-monoenoic phosphatidylcholines under study are summarized
in Table 1.
Figure 2:
Representative DSC heating thermograms for
aqueous dispersions of two series of cis-monoenoic
phosphatidylcholines, C(20):C(18:1)PC and
C(22):C(18:1
)PC with n = 6,
7, 9, 11, 12, and 13. Each aqueous sample contains 3-6
mM lipid, 5 mM phosphate buffer (pH 7.4), 1 mM EDTA, and 50 mM NaCl. Scan rate: 15 °C/h. All
thermograms were obtained from the second or third DSC heating
scans.
The T values of the aqueous
dispersions of C(20):C(18:1
)PC and
C(22):C(18:1
)PC are plotted in Fig. 3against
the position of the cis double bond (
). The
calorimetric data of C(18):C(18:1
)PC reported earlier
from this laboratory (4) are also included in the same plot.
Structurally, these three series of monoenoic lipids differ in their sn-1 acyl chain lengths. Several distinct features are
immediately evident from this plot. First, the parabolic T
-
curve shifts to higher
temperatures as the sn-1 acyl chain length increases. Second,
the
point corresponding to the minimal T
in each curve is up-shifted slightly as the sn-1 acyl chain length increases. In fact, the value of
corresponding to the minimal T
is calculated by the least-squares analysis to occur at n = 10, 10.4, and 10.8 as the sn-1 acyl chain length
increases from 18 to 20 and then to 22, respectively. In each of these
least-squares analyses, an equation of T
= a
+ a
(
)
+ a
(
)
has been
used to fit the experimental date, where a
, a
, and a
are the
coefficients. In addition, the difference in T
between any pair of the three curves
(
T
) shown in Fig. 3decreases as the
-bond migrates from C(10) toward either the carboxyl or the methyl
end. Interestingly, the decrease in
T
is more
pronounced toward the chain methyl terminus. Finally, it is evident
from Fig. 3that all experimental T
values
for monoenoic lipids with the
-bond at C(13) are virtually
superimposable. The
H values for these lipids are, within
experimental errors, also virtually identical (Table 1). The
nearly identical values of both T
and
H indicate that the energy contents of bilayers of
C(18):C(18:1
)PC, C(20):C(18:1
)PC,
and C(22):C(18:1
)PC are, at T < T
, indistinguishable, although these gel-state
bilayers have different thickness due to the variation in the sn-1 acyl chain length.
Figure 3:
The Tversus
for three series of cis-monoenoic
phosphatidylcholines. The experimental data of the
C(20):C(18:1
)PC and
C(22):C(18:1
)PC series are taken from Fig. 2, those of C(18):C(18:1
)PC being
derived from (4) . All three lines connecting the experimental
data are the least-squares lines of a parabolic
function.
In Fig. 4A, the plot of T as a
function of the total number of carbon atoms in the sn-2 acyl
chain (Y) is presented. Within the narrow range of Y from 18 to 22; the T
is observed to be a
linear function of Y for all three series of cis-monoenoic lipids with constant values of X and n. The sn-2 acyl chain of a monoenoic lipid in the
gel-state bilayer can be considered to consist of two linear segments
separated by the cis double bond (vide post). As the Y value increases, the sn-2 acyl chain length of the
lipid molecule in the gel-state bilayer increases; in particular, only
the lower or the shorter segment of the sn-2 acyl chain
increases due to the fixed position of
-bond at C(13). In
addition, the thickness of the hydrocarbon core of the trans-bilayer dimer (N) and the effective chain
length difference between the two acyl chains (
C) also
change as the Y values increase in these cis-monoenoic lipids. The quantitative definitions of N and
C in terms of X, Y, and n for cis-monoenoic phosphatidylcholines are given in the
next section; nevertheless, the data shown in Fig. 4A indicate that the net effect of the length of the shorter segment
of the monounsaturated sn-2 acyl chain, the bilayer thickness,
and the acyl chain length difference can result in a situation in which
the T
value of the lipid bilayer is linearly
related to Y.
Figure 4:
The Tversus the number of carbon atoms in the sn-1 and sn-2 acyl chains for three series of
C(X:C(Y:1
)PC. A, the
number of carbons in the sn-1 acyl chain is fixed in each
series of the cis-monoenoic phosphatidylcholine. B,
the number of carbons in the sn-2 acyl chain is fixed in each
series.
The same nine experimental T values shown in Fig. 4A are replotted in Fig. 4B as a function of the number of carbons in the sn-1 acyl chain (X). It should be noted that the
increase in X from 18 to 20 and then to 22 for the three
series of cis-monoenoic lipids shown in Fig. 4B results in a corresponding increase of two C-C bond lengths
in both
C and N values. Here, each curve in Fig. 4B reflects the subtle change in the net effect of
the simultaneous increase in
C and N on T
, which varies from one lipid series to the next.
Based on the experimental curves illustrated in Fig. 4, A and B, it is evident that the T of
the gel-to-liquid crystalline phase transition for cis-monoenoic phospholipids can be influenced by X, Y, and
, which, in turn, can be related to
the structural parameters
C, N, and the length
of the shorter segment of the sn-2 monounsaturated acyl chain.
The unique value of T
for bilayers of a given cis-monoenoic phosphatidylcholine can thus be considered as a
net result of the fine interplay of the three structural parameters.
Three representative
energy-minimized structures of C(20):C(18:1)PC,
C(20):C(20:1
)PC, and
C(20):C(22:1
)PC are graphically illustrated in Fig. 5. Here, lipids in both the monomeric and dimeric states
are orientated in the same manner with the zigzag plane of the
all-trans sn-1 acyl chain lying on the paper (or x-y)
plane. The sn-2 acyl chain of each phospholipid molecule is
seen to contain two linear segments separated by a
-bond-containing sequence, g
s
s
,
where g
and s
are gauche (+) and skew (+) conformations,
respectively. The zigzag planes of the two segments in the sn-2 acyl chain align perpendicularly to the paper plane;
however, the long axes of the two segments run in parallel with the
directionality of the sn-1 acyl chain.
Figure 5:
The
energy-minimized structures of some representative cis-monoenoic phosphatidylcholines. A, a monomer of
C(20):C(20:1)PC. The total numbers of carbons in the sn-1 and sn-2 acyl chains are identical; hence, the
effective chain length difference between these two acyl chains
projected on the long molecular axis is defined as
C
. In this packing motif, the sn-2
acyl chain has a sequence g
s
s
around the cis double bond. In this graphics display,
the zigzag planes of the two segments of the chain separated by the
sequences g
s
s
are oriented perpendicularly to the zigzag plane of the sn-1 acyl chain. The most striking feature of this packing
motif is the complementary van der Waals' interactions between
the all-trans sn-1 acyl chain and the two segments of the sn-2 acyl chain. The length of the upper segment starting from
the point corresponding to the carbonyl oxygen position of the sn-1 acyl chain is designated as US, while the length of the
lower segment starting from the olefinic C(n+1) carbon is
designated as LS. The relationship between US (or LS) and other
structural parameters is defined in the text and is shown in A. B, the trans-bilayer dimer of
C(20):C(18:1
)PC. The effective chain length
difference between the sn-1 and sn-2 acyl chains
within the monomer is the structural parameter
C. The
distance along the long molecular axis separating the two carbonyl
oxygens in the two opposing sn-1 acyl chains is the thickness
of the hydrophobic core (N), another structural parameter. C, the trans-bilayer dimer of
C(20):C(20:1
)PC. D, the trans-bilayer dimer of C(20):C(22:1
)PC. Note
that the
C and N values vary in (B-D), although the
is fixed at C(13) in the sn-2 acyl chain for all
lipid species.
If we take the chain
length difference between the sn-1 and sn-2 acyl
chains of C(20):C(20:1)PC, shown in Fig. 5A, as
C
, where the
subscript ``ref'' denotes the reference state, then the
C values for mixed-chain
C(20):C(18:1
)PC and C(20):C(22:1
)PC
can be easily identified from Fig. 5, B-D to be (
C
+ 2) and
(
C
- 2), respectively, in terms of
C-C bond lengths along the long chain axis. In fact, the value of
C for a mixed-chain
C(X):C(Y:1
)PC can be generalized as
follows:
C = X - Y +
C
. Furthermore, the chain length of the sn-1 acyl chain is X - 1 carbon-carbon bond
lengths, and the chain length of the monounsaturated sn-2 acyl
chain, in terms of C-C bond lengths, from the point corresponding
to the carbonyl carbon of the sn-1 acyl chain to the methyl
terminus is X - 1 -
C = X - 1 - (X - Y +
C
) = Y - 1 -
C
. It is evident from Fig. 5A that this linear portion of the sn-2 acyl chain can be
considered to consist of two segments separated by the
-bond. The
length of the lower segment (LS), which extends from the olefinic
carbon C(n + 1) to the terminal methyl carbon is (Y - n - 1), and the length of the upper segment
(US) is Y - 1 -
C
- (Y - n - 1) = n -
C
. Finally, let us define the
thickness of the hydrocarbon core of the trans-bilayer dimer (N), which is taken to be the length separated by the two
carbonyl oxygens of the sn-1 acyl chains along the long chain
axis. The value of N can be related to X and Y as follows: N = (X - 1) + VDW
+ (Y - 1 -
C
)
= X + Y + 1 -
C
, where VDW is the van der Waals'
distance between the two opposing terminal methyl groups from the sn-1 and sn-2 acyl chains and is taken to be 3
C-C bond lengths.
For saturated identical-chain
phosphatidylcholines such as C(14):C(14)PC packed in the gel-state
bilayer, the sn-1 acyl chain is effectively longer than the sn-2 acyl chain along the long molecular axis. In fact, the
methyl groups of sn-1 and sn-2 acyl chains within the
same lipid molecule in the gel-state bilayer are separated from each
other by about 1.5 C-C bond lengths(11) . In the presence
of a cis C-C double bond, the sn-2 acyl chain
is further shortened by about 0.8 C-C bond lengths when a
sequence gs
s
is taken into consideration(2, 4) .
Consequently, the value of
C
can be
reasonably assumed to be 2.3 C-C bond lengths for
C(X):C(X:1
)PC packed in the
gel-state bilayer. The various structural parameters for
C(X):C(Y:1
)PC packed in the
gel-state bilayer involving the
C
term, as
discussed in the above paragraph, can thus be expressed as follows:
C = X - Y + 2.3; US
= n - 2.3, and N = X + Y - 1.3. All of these terms have the unit of
C-C bond lengths. The calculated values of the various structural
parameters for all the cis-monoenoic lipids under study are
summarized in Table 2.
It is well known that the gel-to-liquid crystalline phase
transition behavior exhibited by fully hydrated phosphatidylcholines is
modulated by many internal factors, most notably the variation in the
chain length and the chemical structure of the hydrocarbon
chain(12) . In addition, the T and
H values of the main phase transition for aqueous
dispersions of monoenoic phosphatidylcholines depend critically on the
position of the cis carbon-carbon double bond
(
) in the sn-2 acyl chain(4) . For
example, a parabolic T
-
curve with the minimal T
at C(11) is
obtained after a single
-bond is introduced into the sn-2
acyl chain at different positions in C(18):C(18)PC. This parabolic
character of the T
-
curve
has been attributed primarily to the preferentially favorable
interactions between the longer linear segment of the sn-2
acyl chain with the neighboring saturated sn-1 acyl chains in
the gel-state bilayer(4) .
In this study, the parabolic
nature of the T -
curve
observed originally for C(18):C(18:1
)PC is confirmed
by C(20):C(20:1
)PC (Fig. 1). In addition, we
have expanded the subclass of the lipid series used in the earlier work
by including synthetic mixed-chain phosphatidylcholines in which the
total number of carbon atoms in the sn-1 acyl chain is
different from that in the sn-2 acyl chain. Our DSC results
shown in Fig. 3indicate that the T
-lowering effect of the
-bond in the sn-2 acyl chain is clearly influenced by the length of the sn-1 acyl chain when the
-bond is located near the center
of the chain. By contrast, the T
-lowering effect
of the
-bond at C(13) is unchanged as the saturated sn-1
acyl chain increases from 18 to 22 carbon atoms (Fig. 3). The
questions of exactly how the increase in the sn-1 chain length
can affect the T
(or
H) of
mixed-chain monounsaturated phosphatidylcholine when the cis
-bond is near the center of the sn-2 acyl chain and
how the effect is abolished when the cis
-bond is at
C(13) are discussed in the following paragraphs.
For
C(X):C(18:1)PC packed in the gel-state
bilayer, the thickness of the hydrocarbon core of the trans-bilayer dimer (N) and the
C value
of the monomeric lipid increase with increasing chain length of the sn-1 acyl chain, since both N and
C values are linearly related to X when Y in
C(X):C(Y:1
)PC is kept constant (N = X + Y - 1.3 and
C = X - Y + 2.3). As
both N and
C values increase simultaneously and
as the
C value is greater than the van der Waals'
distance separating the two opposing methyl groups near the bilayer
center, the chain methyl ends of two sn-1 acyl chains in the trans-bilayer dimer will interact laterally, at T < T
, giving rise to a more stable bilayer
structure. For instance, as C(18):C(18:1
)PC is
lengthened to C(20):C(18:1
)PC, the N and
C values are each increased by two C-C bond
lengths. The van der Waals' distance between two methyl groups is
4.0 Å or 3.0 C-C bond lengths; consequently, the two sn-1 acyl chains of the trans-bilayer
C(20):C(18:1
)PC are laterally overlapped by about 1.3
C-C bond lengths, which can give rise to an additional favorable
van der Waals' energy that is absent in the trans-bilayer dimer of C(18):C(18:1
)PC with a
smaller N value. As a result, between the
C(20):C(18:1
)PC and the
C(18):C(18:1
)PC bilayers, the T
and
H values associated with the gel-to-liquid
crystalline phase transition are expected to be higher for
C(20):C(18:1
)PC. Likewise, the T
(or
H) value of C(22):C(18:1
)PC is
also expected to be higher than that of
C(20):C(18:1
)PC, since both the N and
C values of the former are 2.0 C-C bond lengths
longer than those of the latter. Experimental data presented in Table 1and Fig. 2show the T
(or
H) for these lipids exhibiting the following order:
C(18):C(18:1
)PC < C(20):C(18:1
)PC
< C(22):C(18:1
)PC. This order is indeed borne out
with our expectation.
It should be reiterated that the presence of a cis double bond in a hydrocarbon chain reduces the rotational
barriers for adjacent C-C single bonds, thus promoting the
conformational variability of the hydrocarbon chain(2) . For a cis -bond located near the center of a hydrocarbon chain
such as the sn-2 acyl chain of
C(18):C(18:1
)PC, the energy barrier for rotating the
C(10)-C(11) bond between two energy minima can be calculated by MM
calculations to be 2.21 kcal/mol. As the
in the sn-2 acyl chain of C(18):C(18:1
)PC is
downshifted from C(9) to C(13) toward the methyl end, the height of the
energy barrier is reduced to 1.88 kcal/mol for rotating the
C(14)-C(15) bond. This leads to a relatively more flexible lower
segment of the sn-2 acyl chain. These MM calculations clearly
show that the flexibility of the chain segment depends on the relative
position of the
-bond. Taking C(18):C(20:1
)PC as
an example, the cis
-bond in this lipid molecule is still
positioned near the center of the linear segment of the sn-2
acyl chain; consequently, rotation about the C(14)-C(15) bond is
restricted somewhat, a situation similar to the rotation about the
C(11)-C(12) bond in the shorter sn-2 acyl chain of
C(18):C(18:1
)PC.
If, at temperatures slightly below
the onset temperature of T, the flexible nature of
the C(14)-C(15) bond in the shorter sn-2 acyl chain of
C(18):C(18:1
) is preserved by and large as the chain
length of the sn-1 acyl chain in
C(18):C(18:1
)PC increases to form new species such as
C(20):C(18:1
)PC and
C(22):C(18:1
)PC, then this rotationally flexible
lower segment of sn-2 acyl chain will perturb laterally the
parallel packing of the lower segment of sn-1 acyl chain
within the same lipid molecule of C(20):C(18:1
)PC or
C(22):C(18:1
)PC. The favorable lateral chain-chain
interaction within the
C region in the crystalline
dimeric unit of C(20):C(18:1
)PC or
C(22):C(18:1
)PC is thus abolished at temperatures
slightly below the T
. The final result is that the T
(or
H) values for
C(18):C(18:1
)PC, C(20):C(18:1
)PC,
and C(22):C(18:1
)PC are virtually identical. It
should be emphasized that the explanation given above is based on the
assumption that the energy barriers for rotating the C(14)-C(15)
bond in the sn-2 acyl chains of
C(18):C(18:1
)PC, C(20):C(18:1
)PC,
and C(22):C(18:1
)PC are nearly identical at
temperatures slightly below T
. In fact, MM
calculations show that the energy barriers for rotating the
C(14)-C(15) bond in the sn-2 acyl chains for these three
monoenoic lipid species are 1.88, 2.21, and 2.31 kcal/mol,
respectively, when these monoenoic lipids are in the highly ordered
conformations. At higher temperatures close to T
,
the lipid chains are more disordered near the methyl ends, and the
differences in the energy barriers for rotating the C(14)-C(15)
bond in the sn-2 acyl chains among these three lipid species
are expected to be further reduced. Our basic assumption stated above
is thus not unreasonable.
Another intriguing question about the
effect of cis -bond on the phase transition behavior of
fully hydrated phosphatidylcholine concerns the strikingly large
reduction in T
. For example, the T
value of the C(22):C(18)PC bilayer is 58.6 °C(13) ,
whereas that of the C(22):C(18:1
)PC bilayer is 16.3
°C (Table 1). Before we offer an answer to this intriguing
question, we need to first identify what structural modifications take
place in the cis-monounsaturated phosphatidylcholines in
comparison with the saturated counterparts and to consider the
difference in T
in relation to the structural
differences.
The basic structure of saturated
C(X):C(Y)PC packed in the gel-state bilayer with a
partially interdigitated motif can be specified by two structural
parameters C and N, each of which is related to X and Y as follows(14) :
C =
X - Y + 1.5
and N = X + Y - 0.5. For
monounsaturated C(X):C(Y:1
)PC packed
in the same motif of gel-state bilayer, the presence of a structural
modification of
-bond in the sn-2 acyl chain
increases the number of structural parameters to four (
C, N, US, and LS). These structural parameters are illustrated in Fig. 5.
Based on the calorimetric results obtained with 50
molecular species of saturated mixed-chain phosphatidylcholines, it has
been demonstrated that the two structural parameters, N and
C, for a given type of saturated mixed-chain
phosphatidylcholine molecules packed in the gel-state bilayer can be
correlated with the T
value of the lipid
bilayer(13, 14) . Specifically, the T
values are related to N and
C as
follows(14) : T
= a
- a
(1/N) - a
(
C/N), where the first
term (a
) in the right-hand side of the equation
corresponds to the extrapolated maximal T
value,
which can be obtained with a lipid bilayer containing an infinitely
large value of N; the second term with a negative sign
indicates that the T
value of a bilayer increases
with increasing values of N; the last term, also with a
negative sign, is regarded as the chain-end perturbation term,
expressed as a normalized
C value, which shows that the
larger the chain-end perturbation, the smaller the T
and that the perturbation becomes insignificant as N
C. More recently, this equation of T
has been refined to include a correction term (13) ;
however, this modified equation does not significantly change our
general interpretation of T
. In summary, the basic
idea underlying this equation is that the T
increases with increasing N and that the T
decreases with increasing
C for
saturated mixed-chain phosphatidylcholines.
When a cis -bond is incorporated into the sn-2 acyl chain of
C(22):C(18)PC at C(13), the N value of the gel-state bilayer
of C(22):C(18:1
)PC, 38.7 C-C bond lengths, is
reduced by 0.8 C-C bond lengths; hence, it is slightly longer
than the N value (38.5) of C(22):C(17)PC. In contrast, the
chain-end perturbation or
C value of
C(22):C(18:1
)PC is smaller than that of C(22):C(17)PC
(6.3 versus 6.5). Interestingly, the T
value of the monoenoic lipid is remarkably smaller than the
saturated one (16.3 °C versus 53.2 °C). Obviously, the
marked difference in T
exhibited by
C(22):C(18:1
)PC and C(22):C(17)PC bilayers cannot be
explained completely by the structural parameters N and
C. Other structural parameters must, therefore, be taken
into consideration when the T
of cis-monoenoic lipids is discussed. In fact, two additional
structure parameters, US and LS, exist for
C(X):C(Y:1
)PC packed in the
gel-state bilayer, each of which can represent the length of the longer
or the shorter segment of the sn-2 acyl chain, depending on
the position of the
-bond. Here, we propose that the entire length
of the shorter segment of the sn-2 acyl chain in the gel-state
bilayer of C(X):C(Y:1
)PC acts as a
structural perturbing element; hence, it is regarded as an important
structural parameter that can modulate effectively the T
of cis-monoenoic lipids. The shorter segment is chosen
because, at temperatures slightly below the T
, it
may have already, at least in part, transformed into a disordered
state.
Of the four structural parameters associated with
C(X):C(Y:1)PC packed in the
gel-state bilayer, three (N,
C, US or N,
C, LS) are independent variables. Since the
shorter segment corresponds to the US when
is less
than C(11) for a C(18):C(18:1
)PC molecule and it is
then switched to the LS as
C(11), we will then
divide the monoenoic C(X):C(Y:1
)PC
into two general groups: group I with a longer upper segment and group
II with a longer lower segment in the sn-2 acyl chain. Within
each general group, lipid molecules with a longer effective saturated sn-1 acyl chain should be treated differently from those with
a longer effective monounsaturated sn-2 acyl chain. This is
due to the recognition that the perturbing effect of the smaller
segment of the sn-2 acyl chain is intrinsically different from
that of
C. In this investigation, all but one of the cis-monounsaturated lipids under study have longer effective sn-1 acyl chains. The discussion will thus focus on those with
longer effective sn-1 acyl chains.
Now, we can proceed to
discuss the relationship between the T and the
three independent structural parameters (N,
C,
and LS) for group I sn-1 saturated/sn-2 cis-monounsaturated phosphatidylcholines. Specifically, we
analyze how the individual contribution of the three structural
parameters affects the T
value, and from these
analyses we can then arrive at a quantitative equation relating T
to all three structural parameters.
First of
all, it is worth noting that the T values
appearing on the right-hand side of each parabolic curve in Fig. 1B and Fig. 3are derived from group I cis-monoenoic phosphatidylcholines that have the same N and
C values but distinctly different LS values. For
example, C(20):C(20:1
)PC and
C(20):C(20:1
)PC of Fig. 1B share the
same common N and
C values of 38.7 and 2.3;
however, their LS values are 8 and 6, respectively (Table 2). The
units for all three structural parameters are the C-C bond
lengths along the long chain axis. It is important to recognize that
all of the T
values exhibited by this group of cis-monoenoic phosphatidylcholines decrease with increases in
LS (Table 2); hence, the structural parameter LS may be regarded
as a perturbing element for the gel-to-liquid crystalline phase
transition. Next, we can identify two pairs of group I cis-monoenoic phosphatidylcholines in Table 2that have
the same common values of
C and LS but different N values; they are
C(20):C(20:1
)PC/C(18):C(18:1
)PC and
C(20):C(18:1
)PC/C(22):C(20:1
)PC.
These two pairs of lipids can serve as examples to demonstrate that the T
increases with increasing N for cis-monoenoic phosphatidylcholines, provided that the other
two structural parameters are held constant. In fact, this
characteristic relationship is well known for saturated identical-chain
phosphatidylcholines, which have the same
C value of 1.5
or a constant ``end effect'' as exemplified by the following
two pairs of lipids: C(20):C(20)PC/C(18):C(18)PC and
C(16):C(16)PC/C(14):C(14)PC. The T
and
H values for the gel-to-liquid crystalline phase transition of these
two pairs of identical-chain phosphatidylcholines increase
progressively with increases in N(15) . For cis-monoenoic phosphatidylcholines with constant values of N and LS but variable
C, two pairs of group I
lipid species,
C(20):C(18:1
)PC/C(18):C(20:1
)PC and
C(22):C(18:1
)PC/C(20):C(20:1
)PC,
can be found in Table 2. The T
values
exhibited by these two pairs of monounsaturated lipids have an inverse
relationship with their
C values, indicating that the
perturbing nature of the end effect increases with increasing
C, leading to a decrease in T
for
the gel-to-liquid crystalline phase transition. A similar reciprocal
relationship between the T
and the
C has also been observed for saturated mixed-chain
phosphatidylcholines with a common value of N such as the
following pairs of positional isomers: C(18):C(16)PC/C(16):C(18)PC and
C(18):C(14)PC/C(14):C(18)PC (16, 17) . Such a
reciprocal relationship, however, applies only to position isomers that
can form, in excess water, partially interdigitated bilayers at T < T
(17) .
Based on the three
relationships discussed above between the T of the
gel-to-liquid crystalline phase transition of a given cis-monoenoic phosphatidylcholine and the underlying three
structural parameters of the given molecule, a general equation can be
formulated for group I sn-1 saturated/sn-2 cis-monounsaturated phosphatidylcholines as follows.
Note that the N value is always greater than LS and
that the sn-1 acyl chain length is defined to be longer than
the length of the sn-2 acyl chain for group I cis-monoenoic phosphatidylcholines packed in the gel-state
bilayer. Consequently, as the value of LS approaches infinity, all the
terms, except a, on the right-hand side of this
equation drop out, leading to T
= a
. In a true physical sense, a
represents the extrapolated T
value of the
gel-to-liquid crystal line phase transition for a unique cis-monoenoic phosphatidylcholine species, which contains two
acyl chains with infinite chain lengths. Thus, a
should be regarded simply as the empirically determined maximal T
value for the gel-to-liquid crystal line phase
transition of cis-monoenoic phosphatidylcholines.
Parenthetically, a
gives such a physical meaning,
only after the ratio of
C/LS, instead of the absolute
value of
C, is used as the last term in this equation. In Table 2, 15 experimental T
values obtained
calorimetrically with samples of group I cis-monoenoic
phosphatidylcholines together with the irrelevant values of structural
parameters are given. After these 15 data sets are substituted into
this common equation, the resulting 15 simultaneous equations can be
subjected to multiple regression analyses to find the most appropriate
coefficients (a
, a
, . . .)
that can statistically best fit all the data sets. We
obtain
with a root mean square error of 0.8441 and a correlation
coefficient () of 0.9940. It should be mentioned that the
extrapolated value of a
is 160.5 °C, which is
very similar to that (162.3 °C) obtained for saturated mixed-chain
phosphatidylcholines(14) . Using , the T
values of various group I cis-monoenoic
phosphatidylcholines can be calculated. The calculated T
for the 15 molecular species that have been used as the input
data are summarized in Table 2. The largest difference between
the experimental and the calculated T
values is
2.1 °C for C(20):C(20:1
)PC, which amounts to a
relative change of 0.7% in Kelvin. This equation also enables us to
calculate the T
values for the following group I
monoenoic lipids: C(18):C(20:1
)PC,
C(22):C(20:1
)PC, C(22):C(24:1
)PC,
and C(24):C(20:1
)PC, of which the experimental T
values are 13.2, 24.5, 41.8, and 24.5 °C,
respectively(4) . The calculated T
values
for the corresponding monoenoic lipids are 13.2, 23.9, 43.1, and 23.9
°C. Again, the agreements between the observed and calculated
values are very good. It should be emphasized that is
obtained statistically based on a collection of rather small data base
of T
values for group I monoenoic lipid with
ranging from 11 to 13 and
C from 0.3 to
6.3; this equation should, therefore, be employed only to estimate the T
values for group I monoenoic lipids with similar
and
C values.
Finally, the 11 data
sets of group II monoenoic phosphatidylcholines summarized in Table 2are subjected to multiple regression analysis using
various combinations of the three structural parameters N,
C, and US. For this group of lipids, the length of the
shorter segment of the sn-2 acyl chain in
C(X):C(Y:1
)PC is specified by the
structural parameter US (Table 2). , given below,
provides an empirical relationship that can correlate satisfactorily
the T
of the gel-to-liquid crystalline phase
transition for group II cis monoenoic phosphatidylcholines
with the underlying structural parameters with root mean square error
= 0.9427 and
= 0.9959.
The empirical contains an additional term in
comparison with , reflecting that the perturbation strength
of the upper segment is less than that of the lower segment. In this
equation, the term C/(
C + US) is a
correction term for
C/US, similar to an equivalent term
used for saturated lipids(13) . However, the basic assumption
that the shorter segment of the sn-2 acyl chain acts as a
structural perturbing element is retained in this equation. Moreover,
the differences between the experimental and calculated T
values, shown in Table 2, for all 11 data
are within 1.5 °C. When is applied to estimate the T
value of
1-palmitoyl-2-oleoyl-phosphatidylcholine, a value of -3.1 °C
is obtained, which is only 0.5 °C smaller than the literature value
of-2.6 °C(6) . Hence, can be considered
as a reasonably good means to estimate the T
value
for group II cis-monoenoic phosphatidylcholines. Taken
together, and can yield T
values of the gel-to-liquid crystal line phase transition for cis-monoenoic phosphatidylcholines that agree closely with the
experimental values (Table 2). This, in turn, can be taken as
evidence to support the postulate that the entire length of the shorter
segment of the sn-2 acyl chain in a cis-monoenoic
phosphatidylcholine molecule can act as a structural perturbing element
in the lipid bilayer at temperatures slightly below T
. It is thus not unreasonable to suggest that the T
(or
H)-lowering effect of a cis
-bondis largely mediated through the shorter segment
of the unsaturated acyl chain. Due to the inherent flexibility of the
C-C bond adjacent to the
-bond, it is most likely that the
basic strength of the perturbation exerted by the shorter chain segment
is considerably larger than that exerted by the chain-end perturbation
or
C. A marked reduction in T
thus
becomes apparent. Moreover, the length of the shorter segment and hence
the strength of the perturbation varies in an inverted V-shaped manner
as a function of
; specifically, the length reaches
the maximum as the
-bond is shifted in position from either end to
the geometric center of the sn-2 acyl chain.This variability
is thus strikingly complementary to the inverted bell-shaped T
profile observed in Fig. 1B and Fig. 3, leading to a coherent picture of the putative role
played by the shorter segment of the unsaturated chain in modulating
the phase transition behavior of the lipid bilayer.