| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Biophys J, March 1999, p. 1228-1240, Vol. 76, No. 3
*Department of Biophysics, Institute of Molecular Biology, Jagiellonian University, Krakow, Poland; #Department of Molecular Science, Research Center, Taisho Pharmaceutical Company Limited, Omiya, Japan; and §Department of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan
 |
ABSTRACT |
|---|
 Top
 Abstract
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
References
|
|---|
Molecular dynamics simulation of the hydrated dimyristoylphosphatidylcholine (DMPC) bilayer membrane in the liquid-crystalline phase was carried out for 5 ns to study the interaction among DMPC headgroups in the membrane/water interface region. The phosphatidylcholine headgroup contains a positively charged choline group and negatively charged phosphate and carbonyl groups, although it is a neutral molecule as a whole. Our previous study (Pasenkiewicz-Gierula, M., Y. Takaoka, H. Miyagawa, K. Kitamura, and A. Kusumi. 1997. J. Phys. Chem. 101:3677-3691) showed the formation of water cross-bridges between negatively charged groups in which a water molecule is simultaneously hydrogen bonded to two DMPC molecules. Water bridges link 76% of DMPC molecules in the membrane. In the present study we show that relatively stable charge associations (charge pairs) are formed between the positively and negatively charged groups of two DMPC molecules. Charge pairs link 93% of DMPC molecules in the membrane. Water bridges and charge pairs together form an extended network of interactions among DMPC headgroups linking 98% of all membrane phospholipids. The average lifetimes of DMPC-DMPC associations via charge pairs, water bridges and both, are at least 730, 1400, and over 1500 ps, respectively. However, these associations are dynamic states and they break and re-form several times during their lifetime.
| |
INTRODUCTION |
|---|
|
Top
Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
References
|
|---|
Phosphatidylcholine (PC) is a most prevalent phospholipid among those that make up eukaryotic cell membranes. It is also a most extensively studied phospholipid in terms of the behavior in model membranes, the dynamics and interaction of alkyl chains in particular. However, those of the PC headgroups in the polar regions of the membrane have been studied much less despite their importance in determining the membrane structure and interactions with molecules in the aqueous phase.
A PC molecule contains groups that are positively and negatively charged, whereas its net electrostatic charge is zero. Representative negatively charged groups of PC include the non-ester phosphate oxygen atoms (Ops) and the carbonyl oxygen atoms in the ester linkages between glycerol and acyl chains (Ocs). The positively charged group is the choline moiety.
Two major classes of short-distance interactions occur between the PC
headgroups. One is the formation of water cross-bridges between
negatively charged groups in which a water molecule is simultaneously
hydrogen (H) bonded to two PC molecules. In our recent molecular
dynamics (MD) simulation study of a PC bilayer membrane
(Pasenkiewicz-Gierula et al., 1997
), we found that ~70% of PC
molecules are cross-linked by H bonded water into clusters of two to
seven molecules. Of 4.5 water molecules H bonded to each
dimyristoylphosphatidylcholine (DMPC) molecule, approximately one, on
average, simultaneously forms H bonds with two oxygen atoms, mainly of
the phosphate and carbonyl groups, of different DMPC molecules and
forms an intermolecular bridge. This result is consistent with
water-mediated interactions between PC oxygen atoms deduced from
experimental results by Nagle (1976), Büldt and Wohlgemuth
(1981), Slater et al. (1993), and Ho et al. (1995). Such interactions
were postulated by Prats et al. (1987), Sakurai and Kawamura (1987),
and Teissie et al. (1990) to explain 20-fold faster lateral transfer of
protons along the membrane surface than in water.
The second class of short-distance interactions that are likely to take
place in the headgroup region in PC membranes involves charge
associations (charge pairs) formed when the oppositely charged groups
are located within 4.0 Å from one another, in analogy to salt bridges
(links) in proteins (Creighton, 1983; Lounnas and Wade, 1997).
In the present study, we mainly addressed this issue. In particular, we pursued further cluster formation of PC molecules caused by charge associations in the DMPC bilayer using MD simulations.
First, we investigated the possibility of formation of charge pairs between a choline methyl group (N-CH3) of one DMPC and non-ester phosphate or carbonyl oxygen atoms (Ops and Ocs, respectively) of another one. In this sense, the present research was an additional development of our previous investigation of the formation of PC clusters based on water cross-bridges. We found that, on average, 42% Ops and 28% Ocs make such charge pairs that link more than 90% of all PC molecules in the membrane.
Experimental studies of Yeagle et al. (1975, 1976, 1977) suggested the
formation of intermolecular charge pairs between the phosphate and the
choline groups of neighboring phospholipid molecules in the
liquid-crystalline bilayers. Although phosphate-choline charge pairs
are also present in the DMPC single crystal, they are exclusively
intramolecular (Hauser et al., 1981). MD simulation of negatively
charged dipalmitoylphosphatidylserine (DPPS) bilayer membrane in the
liquid-crystalline state showed charge interaction between the ammonium
group of one DPPS molecule and oxygen atoms of adjacent DPPS molecules
(Lopez Cascales et al., 1996).
In addition to identifying the short-distance PC-PC interactions in
the membrane, we examined the lifetimes of the water bridges as well as
of the choline-phosphate and the choline-carbonyl charge pairs. The
average lifetime of water bridging of two DMPC oxygen atoms is about 50 ps. The average lifetime of a DMPC-DMPC pair linked by water molecules
is ~730 ps, indicating a fast exchange of H bonds and water bridges,
as postulated by Prats et al. (1987) and Teissie et al. (1990). The
average lifetimes of Op-N-CH3 and Oc-N-CH3 charge pairs are 140 and 174 ps,
respectively. For a DMPC-DMPC pair linked by charge pairs, the average
lifetime is 1416 ps. This indicates the presence of multiple charge
pairs between DMPC molecules in most cases.
DMPC-DMPC associations linked by either or both of short-distance interactions, show higher stability. The lower limit of their average lifetime is 1500 ps, 24% of the association live longer than 3100 ps, the time window of the present analysis.
In this study, we show that 98% of DMPC molecules in the liquid-crystalline bilayer are linked via water bridges and/or choline-phosphate and/or choline-carbonyl charge pairs to form long-lived clusters. This result suggests that headgroup-headgroup interactions greatly contribute to the stability of the membrane structure.
| |
METHODS |
|---|
|
Top
Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
References
|
|---|
Simulation system
The membrane consisting of 72 (6 × 6 × 2) DMPC
molecules was hydrated with 1622 water molecules and simulated for 5100 ps using AMBER 4.0 (Pearlman et al., 1991). There are ~23 water
molecules/DMPC (~38% by weight), which is thought to be sufficient
to fully hydrate the membrane (Gawrisch et al., 1978; Arnold et al.,
1983; Nagle, 1993; Salsbury et al., 1972; Rand and Parsegian, 1989).
The starting configuration was the minimized structure of Vanderkooi
(1991) in the second arrangement. The system was equilibrated for 1100 ps. Details concerning the membrane construction and equilibration have
been described in Pasenkiewicz-Gierula et al. (1997). Fig. 1 shows the structure and numbering of
atoms in the DMPC molecule.
|
Simulation parameters
For DMPC, optimized potentials for liquid simulations (OPLS)
parameters (Jorgensen and Tirado-Rives, 1988) were used. In the original OPLS set, the stretching, bending, and torsion parameters for
the ester group were missing. These parameters were transferred from
the MM3 set (Alinger et al., 1989) with the help of two calibration curves for the stretching and bending force constants. The bond angles
and force constants for the choline group were set in analogy to
OPLS parameters for lysine. The van der Waals parameters for the
phosphorus atom were calculated based on the AMBER parameters and then
re-adjusted to satisfy the proportion (O/N
S/P) between the van der
Waals parameters for the oxygen (O), nitrogen (N), sulfur (S), and
phosphorus (P) atoms. For water, TIP3P parameters (Jorgensen et al.,
1983) were used. The united atom approximation was applied to the DMPC
molecule to reduce computation time. The atomic charges of the DMPC
molecule used in this simulation were taken from Charifson et al.
(1990) except for the first 800 ps of the simulation. During the
initial 800 ps, the charges used were those calculated in
Pasenkiewicz-Gierula et al. (1997) in which the choline and carbonyl
charges were similar to those from Charifson et al. (1990), whereas the
charges for the phosphate group atoms were 10-20% lower. The dipole
moment of the DMPC headgroup calculated for the charge distribution
from Charifson et al. (1990) was 20.4 ± 2.8 Debye and was lower
than that calculated for the charge distribution from
Pasenkiewicz-Gierula et al. (1997) of 26.6 ± 3.3 Debye (both
averaged over 500 ps between 1100 and 1600 ps). The value for the
dipole moment of the DMPC headgroup given in the literature is ~20
Debye (Shepherd and Büldt, 1978; Scott and Lee, 1980; Büldt
and Wohlgemuth, 1981; Frischleder and Peinel, 1982; Bowen and Lewis,
1983; Taylor et al., 1990). For this reason, MD simulation from 800 ps
onward was carried out using atomic charges of Charifson et al. (1990).
Simulation conditions
Three-dimensional periodic boundary conditions using the usual
minimum image convention were used. The SHAKE algorithm (Ryckaert et
al., 1977) was used to preserve the bond lengths of the water molecule,
and the time step was set at 2 fs (Egberts et al., 1994). For nonbonded
interactions, a residue-based cutoff was used with a cutoff distance of
12 Å. To reduce calculation time of nonbonded interactions, each DMPC
was divided into six residues. Each residue was chosen in such a way
that the total electrostatic charge on the residue was close to zero
and the integrity of its chemical groups was preserved
(Pasenkiewicz-Gierula et al., 1997). The list of nonbonded pairs was
updated every 50 steps.
Simulation was carried out at a constant pressure (1 atm) and a
constant temperature (310 K = 37°C), which is above the main phase transition temperature for the DMPC bilayer (~23°C).
Temperatures of the solute and solvent were controlled independently.
Both the temperatures and pressure of the system were controlled by the
Berendsen method (Berendsen et al., 1984). The relaxation times for
temperatures and pressure were set at 0.4 and 0.6 ps, respectively.
Applied pressure was controlled anisotropically, where each direction
was treated independently and the trace of the pressure tensor was kept
constant (1 atm).
| |
RESULTS |
|---|
|
Top
Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
References
|
|---|
Characterization of the membrane system and comparison with experimental data
Fig. 2 shows the time profiles of
the simulation box dimensions (Fig. 2 a), surface area/DMPC
(Fig. 2 b), number of gauche conformations/myristoyl chain (Fig. 2 c), and surface
tension (Fig. 2 d), from the onset of simulation
until 5100 ps. Initial changes in the system dimensions (Fig. 2
a) are caused by rescaling, temporary changes of the
temperature, and addition of another 600 water molecules (cf.
Pasenkiewicz-Gierula et al., 1997). At the beginning of the
equilibration process, the temperature of the system was raised to 550 K for 20 ps, which was effective in breaking the initial crystalline
structure. The temperature was then slowly lowered to 310 K and kept
constant. The potential energy of the system became stable after 500 ps
of the simulation time. Approach toward a thermally equilibrated DMPC
bilayer in the liquid-crystalline phase has been described in detail
(Pasenkiewicz-Gierula et al., 1997). In that paper we concluded that
the membrane had reached thermal equilibrium after 1100 ps of MD
simulation.
|
Between 1100 and 5100 ps of MD simulation, the average surface area per
DMPC of 60.2 ± 1.0 Å2, the average number
of gauche rotamers/myristoyl chain of 2.8 ± 0.1, and
the nearly zero average tilt angle of hydrocarbon chains with respect
to the bilayer normal were obtained, which is consistent with
experimental observations (Nagle et al., 1996; Nagle, 1993; Casal and
McElhaney, 1990; Moser et al., 1989; Meier et al., 1982), theoretical
calculations (Carlson and Sethna, 1987), and other simulations (Tu et
al., 1995). The electron density profile of the bilayer and the profile
of the order parameter are in agreement with experimental data (Levine
and Wilkins, 1971; Hubbell and McConnell, 1971).
The surface tension in the simulation box was monitored from ~800 ps
of the simulation time. After equilibration, the average surface
tension (Zhang et al., 1995) is 
0.6 ± 123 dyn/cm (Fig. 2
d). Its nearly zero value is in good agreement with
theoretical predictions of Jähnig (1996) and MD simulation of Tu
et al. (1996). Large fluctuations of this parameter are caused by large
fluctuations in the system pressure.
Fig. 3 a shows the distribution of the orientation of the P-N vector with respect to the membrane normal (the z axis). The distribution of the inclination angle is broad and almost uniform in the range of angles between 0 and 100°.
|
Orientations and conformations of the DMPC headgroups in the
liquid-crystalline bilayer significantly differ from those in the
initial structure. Fig. 3 b shows the distribution of angles between the P-N vector and the in-plane x axis in the
initial structure and in the liquid-crystalline bilayer at 5100 ps
(calculated for last 300 ps, i.e., between 4800 and 5100 ps). In the
former case, the vector has only four orientations (57°, 64°,
124°, and 119° for DMPC A and B in the upper and lower leaflet,
respectively). In the latter case, the angles span the whole range from
180° to 180°.
These results suggest that the simulated membrane obtained here is well equilibrated, stable over a long period of time, and reproduces various properties of PC bilayers in the liquid-crystalline phase that have been observed experimentally. Therefore, it is concluded that this membrane provides a good model for DMPC membranes, and that by analyzing it at the level of individual atoms, we can obtain experimentally inaccessible information about structure and dynamics of PC membranes.
In the present report, in analyzing trajectories of 4.0 ns MD simulation after equilibration, we concentrate on formation of charge pairs between positively charged choline methyl groups and negatively charged oxygen atoms of the phosphate or carbonyl groups in the membrane, which along with bridging by hydrogen bonded water molecules leads to formation of extended DMPC clusters. We also estimate the lifetimes for water bridging and charge pairing.
DMPC-DMPC association via charge pairs and water bridges
Interactions between Op and N-CH3
The radial distribution function (RDF) of the phosphorus atoms relative to a nitrogen atom, gP-N, (intramolecular distances are not included) has a distinct maximum at 4.8 Å (Fig. 4 a). The presence of the maximum indicates that in the membrane the choline and the phosphate groups stay in a close, preferred distance from one another. For comparison, the RDFs of Ps relative to P and Ns relative to N are also shown in Fig. 4, c and d, respectively. These functions have broader peaks at longer distances (when an intermolecular Op-Op water bridge is made, the average P-P distance is 6.65 ± 1.41 Å).
|
0.9 in units of an
electronic charge (e)) and CH3 (a united atom C3
with a positive charge of 0.14 (e) that contribute to the peak in RDF
at 3.4 Å must be strongly interacting electrostatically with each
other and form transient Op-N-CH3 charge pairs.
|
). These interactions contribute to protein stability
to different extent (Honig and Hubbell, 1984; Lounnas and Wade, 1997).
By analogy to the definition of salt links, introduced by Lounnas and
Wade (1997) to assess their contribution to cytochrome P450cam
stability, we call a pair of Op and N-CH3, which
are located within 4.0 Å from each other (which is less than the
position of the first minimum in the RDF of 4.6 Å) a charge pair. A
sphere of radius 4.0 Å centered on N-CH3 is
called here an interaction sphere.
As can be seen in Fig. 5, a and c, the RDFs of
Ops and the water oxygen atoms, Ows, relative to a choline methyl group
are very similar, particularly in the positions of the major peaks (~3.5 Å) and the first minimum (~4.6 Å). Thus, the interaction spheres around N-CH3 for Ops and Ows coincide,
which is consistent with our finding that Op, Ow, and
N-CH3 moieties mutually interact and compete
(results will be published elsewhere).
On average, 61 Ops are located inside the interaction spheres of 216 N-CH3, which constitutes 42% of all 144 Ops in
the membrane (Table 1). For comparison,
503 water molecules are located inside the interaction spheres of
N-CH3s (31% of all water molecules in the
membrane). Of them, 143 are simultaneously H bonded to Ops and 360 interact solely with the N-CH3 groups.
|
|
|
|
Interactions between Oc and N-CH3
The RDF of the carbonyl carbon atoms relative to a nitrogen atom (intramolecular distances are not included), gC-N, is shown in Fig. 4 b. It has a distinct maximum at 5.0 Å. As one can see in Fig. 5 b and read from Table 1, the percentage of Ocs that make charge pairs with N-CH3 in the membrane is smaller than that of Ops (28 vs. 42%). Of all intermolecular charge pairs N-CH3 forms, 60% are with Ops and 40% are with Ocs. However, Ocs make intramolecular charge pairs more frequently (in 22% of cases) than Ops (in 3% of cases), but intramolecular charge pairs constitute merely 12% of all charge pairs formed in the membrane. The average distance between Oc and N-CH3 comprising of an intermolecular charge pair is 3.52 ± 0.26 Å and is almost the same as that between Op and N-CH3 (Table 2). An example of an Oc that simultaneously forms charge pairs with two N-CH3s is given in Fig. 6 b. An example of intramolecular Oc-N-CH3 charge pair is given in Fig. 6 d. The snapshots of Oc-N-CH3 that lasted for at least 60% of the 10 ps period at the time of 2000 ps and of 3500 ps, are shown in Fig. 8.
|
DMPC-DMPC interactions via charge pairs
The average number of all intermolecular charge pairs (Op-N-CH3 and Oc-N-CH3) in the membrane built of 72 DMPC molecules is 150, whereas the number of DMPC molecules linked via charge pairs is 67, and they constitute 93% of all DMPC molecules in the membrane (Table 3). The average number of all intramolecular charge pairs in the membrane is 20 (Table 1).
|
DMPC-DMPC interactions via water bridges
Intermolecular water bridges between DMPC molecules in each layer of the membrane at two time points separated by 1500 ps (2000 and 3500 ps) are shown in Fig. 9. The water bridges that, during 10 ps (2000-2010 ps and 3490-3500 ps), lasted for at least 60% of the time are drawn with a solid line and the ones that lasted for 40-60% of the time are drawn with a dash line. The ones that lasted for less than 40% of the time are not shown in the figure. This procedure followed from the fact that bridges can temporarily break. A double (triple) line indicates a double (triple) bridge. The location of the headgroup is represented by that of the phosphorus (P) atom. The display of intermolecular bridges under periodic boundary conditions requires different arrangements of the P atoms at the two time points. In the figure, the DMPC oxygen atoms involved in water bridging are not discriminated, i.e., bridges linking phosphate nonester and carbonyl oxygen atoms (Op-Oc bridges) are marked in the same way as Op-Op bridges.
|
) in which 70% of DMPC
molecules were linked into clusters of 2 to 7 DMPC molecules. However,
previous results were obtained for much shorter MD simulation;
apparently, re-arrangement of DMPC headgroups to participate in the
cluster formation is a lengthy process, the rotational correlation time
of the P-N vector is 3.7 ns (Pasenkiewicz-Gierula and Róg, 1997).
The time dependence of water bridging is discussed below.
DMPC-DMPC interactions via charge pairs and/or water bridges
The average number of DMPC-DMPC pairs formed via charge pairs and/or water bridges in the 72 DMPC molecule membrane is 97, whereas the number of DMPC molecules linked via these short-distance interactions is 71 and they constitute 98% of all DMPC molecules in the membrane (Table 3). The snapshots of DMPC-DMPC links via water bridges and/or charge pairs that lasted for at least 60% of the 10 ps period at the time of 2000 ps and of 3500 ps are shown in Fig. 10.
|
Lifetime of DMPC-DMPC associations
The above analysis indicates that in a fully hydrated phospholipid bilayer membrane, at a given time, the headgroups of DMPC molecules extensively interact with one another via water bridges and/or charge pairs. Here we describe the lifetimes of these interactions within the time window of 3100 ps, between 2000 and 5100 ps of the simulation.
Lifetime of charge pairs between headgroups
As described previously, in the liquid-crystalline state, charge pairs are formed between a nonester phosphate oxygen, Op, and a methyl group of the choline residue, N-CH3, and between a carbonyl oxygen, Oc, and N-CH3. The average number of intermolecular Op-N-CH3 pairs in the present membrane is 90 and of Oc-N-CH3 pairs is 60 (Table 1) (the number of Ops, Ocs, and N-CH3 in the membrane is 144, 144, and 216, respectively
we do not
discriminate between O13 and O14 and between O22
and O32). The average number of DMPC-DMPC charge pairs, linked via
either Op-N-CH3 pairs or
Oc-N-CH3 pairs, is 67 (Table 3).
Op-N-CH3 charge pairs. To calculate the
lifetime, a history of each charge pair, formed during a period between
2000 and 4000 ps, was monitored for the time from its first appearance till the final time of 5100 ps, every 1.0 ps. In each case it was
established which Op and N-CH3 of two DMPC were
charge paired. In this analysis, if a pair was temporarily broken but
re-formed within 60 ps between the same pair of Op and
N-CH3, the break was ignored, whereas a break
longer than 60 ps was treated as the final decay of the pair. The
limiting value of 60 ps was chosen because 60 ps was the time of the
longest firm pairing (binding without breakage) between breaks.
Besides, occurrence of breaks longer than 60 ps was below 6%. The same
cutoff time of 60 ps for breaks is used in the analyses of charge
pairing and water bridging described below.
Fig. 11 a shows the
distribution of the lifetimes of Op-N-CH3
pairing. On average, an Op-N-CH3 charge pair
lives for 140 ± 225 ps, during which it goes from on to off to on
pairing state 18 times. The frequency of breaks is ~1.3 × 1011 per second. The term, firm pairing, was used
for bonding without breakage (bonding between breaks, but only observed
every 1 ps). The average firm pairing time of
Op-N-CH3 charge pair is 3.4 ps, and the average
temporary break time is 3.8 ps (Table 4).
|
|
Lifetime of DMPC-DMPC associations via water bridges
In this paragraph, the lifetimes of water bridges formed between phosphate nonester and carbonyl oxygen atoms and the lifetime of DMPC-DMPC bridged pairs are given. The average number of H bonded water molecules in the membrane is 323 (4.5/DMPC) and, on average, 64 of them (~20%) make bridges (Pasenkiewicz-Gierula et al., 1997). The
average number of DMPC-DMPC pairs bridged by water is 40 (Table 3).
Water bridges. The lifetime of water bridges was
determined in a similar way as the lifetime of charge pairs. During a
period between 2000 and 4000 ps, a history of each water bridge was
monitored for the time from its first appearance till the final time of
5100 ps, every 1.0 ps. In each case it was established which oxygen
atoms of two DMPC were bridged by a given water molecule. The term,
firm bridging, was used for bonding between breaks (but only observed
every 1 ps). Consistent with the analysis of charge pairs, a
break longer than 60 ps was treated as the final decay of the bridge.
Fig. 12 a shows the
distribution of bridging lifetimes. The average lifetime is 49 ± 49 ps.
|
)). A bridge is finally broken when the water molecule has
been away from at least one of its original pair-oxygens for a time
longer than 60 ps, i.e., makes an H bond with only one or none of the
oxygens it originally bridged. Water bridging seems a less dynamic
state than charge pairing, probably because a water molecule, because
of its much less limited mobility, easily diffuses away from its H
bonding partner. Thus, both the number of breaks and the average
lifetime of H bonds are much smaller than those of charge pairs.
DMPC-DMPC water-bridged pairs. At the time of 2000 ps
all DMPC-DMPC pairs that were bridged by water molecules were selected
(the pool of DMPC-DMPC bridged pairs, 40 pairs (Table 3)). It was also
established which DMPC molecules make each pair, but exchanges of water
molecules bridging the pair were allowed. Over the time of 3100 ps,
every 1.0 ps a fraction of DMPC-DMPC bridged pairs remaining in the
pool was determined.
Fig. 12 d shows the distribution of the lifetimes of
DMPC-DMPC bridged pairs. As for water bridges, the lifetime was
defined as the time after which a DMPC-DMPC pair is broken for a time longer than 60 ps. Approximately 7.5% of the DMPC-DMPC bridged pairs
lives longer than 3100 ps, the time window of the present analysis.
This allows only to estimate the lower limit for the average
lifetime of a DMPC-DMPC bridged pair, which is 730 ± 887 ps
(Table 4).
The average time of firm bridging, temporary break time, and the
number and frequency of breaks are given in Table 4.
A much longer average lifetime of DMPC-DMPC pairs bridged by water
(730 ps) than water bridges (49 ps) indicates that although bridging
bonds may decay relatively fast, in their place other ones are formed
either through water or bonds exchange. In effect, several headgroups
of DMPC remain water bridged for times longer than 1 ns. The
frequencies of breaks of DMPC-DMPC pairs bridged by water and by
charge pairs are practically the same and equal to 0.7 × 1011/s.
Lifetime of DMPC-DMPC links via charge pairs and/or water bridges
All DMPC-DMPC links via charge pairs and water bridges were found at the time of 2000 ps (the pool of 97 DMPC-DMPC links (Table 3)), and the lifetime analysis of the links was carried out as described above. Fig. 13 shows the distribution of the lifetimes of DMPC-DMPC links. Approximately 24% of them stayed longer than 3100 ps, the time window of the present analysis. This only allows estimation of the lower limit of their average lifetime, which is 1500 ± 1165 ps (Table 4). This value is only slightly greater than that for charge pairing or water bridging probably because of the insufficient length of the trajectory.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
References
|
|---|
Headgroup-headgroup interactions: structural properties
Our previous work (Pasenkiewicz-Gierula et al., 1997) indicated
that 76% of DMPC molecules in the bilayer are linked by single or
multiple water bridges. In the present research we showed that DMPC
headgroups also interact directly via Coulombic attraction between
positively charged choline methyl groups of one DMPC molecule and
negatively charged phosphate or carbonyl oxygen atoms of another molecule. Out of 170 charge pairs present in the bilayer at any instant, 150 (88%) are intermolecular and 20 (12%) are
intramolecular. The intermolecular charge pairs link 93% of the DMPC
molecules into clusters of varying sizes.
Water bridges and charge pairs form an extended network of interactions among PC headgroups. These interactions link 98% of all PC molecules in the membrane; on average, merely one PC molecule is not linked to the remaining ones.
In the present study, we used the atomic charges established by
Charifson et al. (1990) and a united atom approximation. These choices
would affect the charge pairing observed in this work, which is
discussed below.
Both in H bonding (water bridging) and charge pairing, the dominant
interaction is the Coulomb attraction, which depends on the charge
distribution on the PC molecule, and surrounding water. We compared
formation of H bonds between PC and water for two charge distributions;
one calculated by us (Pasenkiewicz-Gierula et al., 1997) and the other
by Charifson et al. (1990) (see Methods). The total numbers of H-bonded
water molecules per DMPC were near 4.5 for both charge distributions,
thus both distributions well reproduced experimental estimates (Arnold
et al., 1983; Gawrisch et al., 1978; Nagle, 1993). But we are unable to
quantitatively estimate the influence of different point charges on
charge pairing. However, because the membrane obtained in this
simulation well reproduced almost all experimental parameters examined
in this series of study, including H bonds with water, we believe that the choice of charges made here is reasonable. Application of the
united atom approximation to the DMPC molecule introduces slightly
different charge distribution on the choline group and neglects a
slightly polar character of the N-CH3 groups.
This might modify to some extent the Coulombic interactions of the choline group. However, both in the all-atom (Alper et al., 1993) and
the united-atom (Pasenkiewicz-Gierula et al., 1997) models, water
orientation around the choline group is similar and typical of the
clathrate structure. Thus, we believe that the united atom approach did
not significantly modify the Coulomb interactions of the choline group.
There have been considerable debates on the proper value for surface tension to be used in membrane simulations. The average surface tension observed in this simulation was ~0 dyn/cm after equilibration, which is consistent with the fact that no surface tension was artificially applied.
Both water bridges and charge pairs are present in the DMPC single
crystal (Hauser et al., 1981), in which 1) all DMPC molecules along one
crystallographic axis are linked via water H bonded to the phosphate
oxygen atoms to form infinite ribbons and 2) all the choline and
phosphate groups form intramolecular charge pairs. However, the
carbonyl groups are involved neither in water bridging nor charge
pairing in the DMPC single crystal.
31P[1H] nuclear
Overhauser effects studies of liquid-crystalline phospholipid bilayers
(Yeagle et al., 1975, 1976, 1977; Yeagle, 1978) demonstrated a strong
interaction between the phosphate group and the methyl groups of the
choline moiety of different lipid molecules. The result was further
supported by the observations that increasing amounts of cholesterol
weakened intermolecular headgroup interactions because of the spacing
effect (Yeagle et al., 1975, 1977).
Headgroup-headgroup interactions: lifetimes
In the liquid-crystalline DMPC bilayer membrane, the average
lifetime of DMPC-DMPC pairs bridged by one or more water molecules is
much longer than that of bridging H bonds (730 vs. 50 ps). This
indicates that bridging water molecules and/or bridging bonds linking
two DMPC molecules are effectively exchanged. This result is in accord
with experimental predictions of Prats et al. (1987), Sakurai and
Kawamura (1987), and Teissie et al. (1990), based on the observation of
20-fold faster lateral transfer of protons along the membrane surface
than in water.
The average lifetimes of the Op-N-CH3 and Oc-N-CH3 charge pairs are about three times longer than that of the water bridges (~170 vs. 50 ps), and the average lifetime of DMPC-DMPC associations via charge pairs is two times longer than that of DMPC-DMPC associations via water bridges (1420 vs. 730 ps). In addition, 93% of DMPC molecules are linked by charge pairs, whereas 76% are linked by water bridges. These suggest that the contribution of the charge pairs to the membrane stability in the liquid-crystalline phase is more significant than that of water bridges. It should be stressed that both water bridges and charge pairs are breaking and re-forming dynamically. The ranges are for firm bonding time from 3.4 to 12.0 ps, for break time from 2.3 to 4.0, and for frequency of breakage from 0.7 × 1011 to 1.3 × 1011 1/s. In the present research, if a water bridge or charge pair temporarily breaks but re-forms within 60 ps between the same pair of DMPC atoms/groups, it is counted as unbroken.
Analysis of DMPC-DMPC associations via charge pairs and/or water bridges shows that headgroups of 71 of 72 DMPC molecules (98%) are linked in a network for an average time of at least 1500 ps. Each individual pair of DMPC molecules is, on average, firmly bonded for 14 ps between 2.6 ps breaks. The longest acceptable break time is 60 ps. The break frequency is 0.6 × 1011/s.
Results presented in this study allow us to postulate that in a PC bilayer membrane PC headgroup-headgroup interactions, both via water bridges and via Coulombic attraction, contribute to the stability of the membrane in the liquid-crystalline phase. These interactions involve 98% of DMPC molecules in the membrane and are stable in the nanosecond time scale.
In the presence of ions, the situation may be quite different. The
charge pairs could be formed by way of ions, as was shown to take place
between cationic surfactant and chloride counterions in a recent MD
simulation study of Tobias and Klein (1996). A systematic study on the
effect of varying concentration of sodium chloride on PC membranes is
underway in our laboratories.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported in part by a grant from The Polish Science Foundation (BIMOL 103/93).
| |
FOOTNOTES |
|---|
Received for publication 31 July 1998 and in final form 23 November 1998.
Address reprint requests to Marta Pasenkiewicz-Gierula, Department of Biophysics, Institute of Molecular Biology, Jagiellonian University, al. Mickiewicza 3, 31-120 Krakow, Poland. Tel.: 48-12-634-2008; Fax: 48-12-633-6907; E-mail: mpg{at}mol.uj.edu.pl
| |
REFERENCES |
|---|
|
Top
Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
References
|
|---|
phase dipalmitoylphosphatidylcholine bilayers.
Biophys. J.
70:1419-1431[Abstract].
Biophys J, March 1999, p. 1228-1240, Vol. 76, No. 3
© 1999 by the Biophysical Society 0006-3495/99/03/1228/13 $2.00
This article has been cited by other articles:
 |
|
 |
|
  S. L. Duncan and R. G. Larson Comparing Experimental and Simulated Pressure-Area Isotherms for DPPC Biophys. J., April 15, 2008; 94(8): 2965 - 2986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Li, M. Yi, J. Hu, H.-X. Zhou, and T. A. Cross Solid-State NMR and MD Simulations of the Antiviral Drug Amantadine Solubilized in DMPC Bilayers Biophys. J., February 15, 2008; 94(4): 1295 - 1302. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Oncins, L. Picas, J. Hernandez-Borrell, S. Garcia-Manyes, and F. Sanz Thermal Response of Langmuir-Blodgett Films of Dipalmitoylphosphatidylcholine Studied by Atomic Force Microscopy and Force Spectroscopy Biophys. J., October 15, 2007; 93(8): 2713 - 2725. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Popova and D. K. Hincha Effects of Cholesterol on Dry Bilayers: Interactions between Phosphatidylcholine Unsaturation and Glycolipid or Free Sugar |
shows the location of the phosphorus atom in the x,
y-plane. A pair of open squares represents images of "real"
PC molecules to show a link between headgroups of adjacent simulation
boxes via periodic boundary conditions.
