Article pubs.acs.org/Langmuir
Cholesterol-Induced Condensing and Disordering Effects on a Rigid Catanionic Bilayer: A Molecular Dynamics Study An-Tsung Kuo and Chien-Hsiang Chang* Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan S Supporting Information *
ABSTRACT: Molecular dynamics simulation is applied to investigate the bilayer properties of a novel catanionic vesicle composed of an ion pair amphiphile, hexadecyltrimethylammonium−dodecylsulfate (HTMA-DS), with cholesterol. Structural properties, such as molecular organization, orientation, and conformation, were analyzed from the resulting trajectory. Simulation results showed that cholesterol could induce both condensing and disordering effects on the rigid HTMA-DS bilayer. The condensing effect of cholesterol was ascribed to the maximizing contact between cholesterol ring and the neighboring hydrocarbon chains. Thus, the inserted cholesterol ring restrained the neighboring hydrocarbon chain segments from motion and increased the order of the neighboring hydrocarbon chains. However, the presence of cholesterol would increase the distance between head groups of HTMA-DS and induce a shift of DS− head groups toward the inside of the bilayer. This led to the protrusion of the HTMA+ head groups and conformational disorder in the front segments of HTMA+ hydrocarbon chains. In addition, the cholesterol-induced void in the hydrophobic core of the HTMA-DS bilayer increased the motion freedom of the terminal segments of the hydrocarbon chains. The cholesterol-induced space in the polar region and void in the nonpolar region of the bilayer led to a conformational disorder. With high cholesterol contents, the conformational disorder effect would overwhelm the condensing effect, resulting in the apparent disordering effect on the rigid HTMA-DS bilayer.
1. INTRODUCTION Recently, mixtures of two oppositely charged surfactants, catanionic mixtures or catanionic surfactants, have attracted a lot of attention because of their potential in pharmaceutical applications.1,2 After removing counterions from the mixtures, the complex composed of two oppositely charged amphiphilic moieties is referred as ion pair amphiphile (IPA).2 Both of catanionic surfactant and IPA can form catanionic vesicle, which has the potential as a drug or DNA carrier.1−4 It has been reported that catanionic vesicles prepared from IPAs with a classic mechanical disruption process usually showed poor physical stability.5,6 To improve the physical stability of catanionic vesicles, additives, such as charged surfactants and cholesterol, were introduced in the preparation of vesicles.7−11 It is believed that additional charged surfactants can assist the formation of charged vesicles and cholesterol additive can adjust the molecular packing of the vesicular bilayers. That is the improved intervesicle or intravesicle interaction would promote the formation of stable vesicles. Cholesterol is an important constituent of animal cell membranes. The roles of cholesterol in the cell membranes include regulation of membrane fluidity,12,13 reduction of passive permeability of small molecules, such as water and gases,14,15 and increase of membrane mechanical strength.16−18 The effects of cholesterol on lipid bilayer characteristics have been widely studied.19−22 Cholesterol may also play an © 2013 American Chemical Society
important role in catanionic vesicular bilayer characteristics. The components of catanionic vesicular bilayers are different from lipid bilayers, and the effects of cholesterol on the molecular packing of catanionic vesicular bilayers are not expected the same as those of lipid bilayers. It is thus necessary to explore the effects of cholesterol on the catanionic vesicular bilayer characteristics in order to fully understand the stabilization mechanisms for catanionic vesicles. The molecular dynamics (MD) simulation is a useful tool for investigating the microscopic structures of vesicular bilayers at the molecular level. A large number of MD studies examining the effects of cholesterol on the lipid bilayer characteristics using fully atomistic as well as coarse-grained descriptions have been reported.22−30 These MD studies have provided insights into bilayer properties, such as bilayer structure,22−27 elastic modulus,28,29 and water permeability,30 of lipid bilayers with cholesterol from a molecular viewpoint. With the aim of elucidating how cholesterol affects the molecular packing characteristics of catanionic vesicular bilayers, a series of MD simulations of a catanionic vesicular bilayer composed of IPA with cholesterol were conducted. Because the physical stability of the catanionic vesicle Received: September 23, 2013 Revised: November 16, 2013 Published: December 17, 2013 55
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was adopted for all MD simulations. All simulations were carried out for 50 ns. The period of first 20 ns is considered the equilibration period, and the results obtained in the following 30 ns period were used to analyze the bilayer structure characteristics.
composed of an IPA, hexadecyltrimethylammonium−dodecylsulfate (HTMA-DS), could be enhanced by the addition of cholesterol,31 HTMA-DS was chosen as a model IPA molecule in this work. Structural properties, such as molecular organization, orientation, and conformation, were then analyzed to elucidate the cholesterol effects on molecular packing of catanionic vesicular bilayers.
3. RESULTS AND DISCUSSION 3.1. Average Area per Molecule. The time evolution of the average area per molecule for the HTMA-DS/cholesterol bilayers with various compositions during the last 30 ns is plotted in Figure 2. The average area per molecule significantly
2. SIMULATION METHODS Molecular structure models of hexadecyltrimethylammonium moiety (HTMA+), dodecylsulfate moiety (DS−), and cholesterol were constructed by using Discovery Studio 3.132 (Figure 1). Before constructing the initial structure model of HTMA-
Figure 2. Time evolution of the averaged molecular areas of HTMADS/cholesterol bilayers.
decreased with the increase in the mole fraction of cholesterol (XChol). This reflects the cholesterol-induced condensation of molecular packing within the catanionic bilayers. To confirm the cholesterol-induced condensing effect, the area per HTMADS molecule listed in Table 1 was calculated by subtracting the Table 1. Averaged Molecular Areas (A), Areas per HTMADS (AHTMA‑DS), and Elastic Area Compressibility Moduli (KA) of HTMA-DS/Cholesterol Bilayers Figure 1. Molecular structures of hexadecyltrimethylammonium moiety (HTMA+), dodecylsulfate moiety (DS−), and cholesterol. An ion pair amphiphile (IPA), HTMA-DS, is formed of HTMA+ and DS−.
DS, the HTMA+ and DS− were placed at an appropriately close distance (distance between N and S was approximately 0.4 nm). Next, Packmol33 was used to randomly build the initial structure of the mixed HTMA-DS/cholesterol bilayer. Each bilayer system was composed of 128 surfactant and 3464 water molecules (Table S1 in Supporting Information). The HTMA+, DS−, and cholesterol molecules were parametrized using the CHARMM PARAM27R force field.34,35 The water molecule was described by the TIP3P model.36 All-atom MD simulations were carried out using Gromacs version 4.53 package.37,38 Periodic boundary conditions were applied in all three directions. The MD simulation was performed with an isothermal−isobaric (NPT) ensemble. The temperature of the simulation system was maintained at 298 K by the Nosé−Hoover chain thermostat,39 and the pressure was controlled at 1 bar by the semi-isotropic Parrinello−Rahman barostat.40,41 Long-range electrostatic potential was calculated using the particle-mesh Ewald (PME) method,42 and Lennard-Jones (LJ) pair potentials were evaluated within a cutoff distance of 1.2 nm with a smooth switching function above 1 nm. Constraints were applied to all bonds using the LINCS algorithm.43 The time step size of 2 fs
XChol
A [nm2]
AHTMA‑DS [nm2]
KA [mN/m]
0 0.09 0.30 0.50
0.415 0.392 0.380 0.378
0.415 0.394 0.384 0.386
1967 2412 9551 6305
total cross-section area of cholesterol molecules from the total surface area of the bilayer. With the calculation, the crystalline cholesterol molecular area of ∼0.37 nm2 was used to evaluate the total cross-section area of cholesterol molecules in the bilayer.44 Table 1 apparently demonstrates the condensing effects of cholesterol on the HTMA-DS bilayer; that is, the area per HTMA-DS molecule decreased with increased XChol. Because the HTMA-DS bilayer is in the gel phase at 298 K,45,46 it is thus interesting to note that cholesterol could induce the condensation of hydrocarbon chains in the rigid HTMA-DS bilayer. The details will be discussed in section 3.5. Figure 2 also shows that fluctuations in the average area per molecule attenuated with increased XChol, which may reflect the less flexible bilayer characteristic induced by cholesterol. To compare the mechanical property of the bilayers, the area compressibility modulus, KA, was calculated by using the relationship derived with the linear response theory47,48 KA = 56
kT ⟨A⟩ N ⟨δA2 ⟩
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Figure 3. Snapshots of the HTMA-DS/cholesterol bilayers during MD simulation: (a) HTMA-DS, (b) XChol = 0.09, (c) XChol = 0.30, and (d) XChol = 0.50. (e) The sketch of the cholesterol and neighboring HTMA-DS molecules in the mixed HTMA-DS/cholesterol bilayer. The substances were colored as follows: (blue) HTMA, (red) DS, (yellow) cholesterol, and (cyan) water. H atoms on the surfactants are not shown.
Figure 4. Probability distributions of selected atoms along the bilayer normal: (a) HTMA-DS, (b) XChol = 0.09, (c) XChol = 0.30, and (d) XChol = 0.50.
HTMA+ and DS− could create a void which allowed the long hydrocarbon chains to reorient in the hydrophobic core. This led to a less tight packing near the slip plane. With the inclusion of cholesterol in the HTMA-DS bilayer, regular molecular arrangements of the mixed bilayers were also observed (Figures 3b−d). It appeared that cholesterol molecules tended to be parallel with neighboring hydrocarbon chains (Figure 3e) and located near the bilayer center. Nevertheless, the void in the bilayer center was more pronounced in the presence of cholesterol, and thus the wobble of the terminal segments of the HTMA-DS hydrocarbon chains became significant. The probability distributions of selected atoms along the bilayer normal (z-axis) are plotted in Figure 4. In the HTMADS bilayer, the nitrogen (N) and sulfur (S) atoms in HTMADS head groups located almost at the same z-position. The peak position of N in the HTMA+ head group was not changed with the presence of cholesterol. However, the peak position of S in the DS− head group was slightly shifted toward the inside of the bilayer and tended to locate between the head groups of HTMA+ and cholesterol when XChol was increased.
where A is the averaged cross-sectional area per molecule and N is the number of molecules aligned in a bilayer leaflet. The angle brackets denote the ensemble average. This equation indicates that the bilayer area compressibility modulus is inversely proportional to area fluctuation. The calculated moduli of mixed HTMA-DS/cholesterol bilayers are listed in Table 1. With the incorporation of cholesterol, the KA value of the mixed bilayer became large and achieved the maximum at XChol = 0.3. Because the higher area compressibility modulus of a bilayer generally corresponds to the higher tensile strength,47 it is suggested that the addition of cholesterol can enhance the stability of the bilayers under applied external mechanical forces. 3.2. Overall Bilayer Structure. Figure 3 displays the snapshot of each bilayer system. A snapshot of the HTMA-DS bilayer is shown in panel a. Hydrocarbon chains of a HTMADS bilayer were in a regular arrangement, except for those near the slip plane between the two bilayer leaflets. The regular arrangement of the hydrocarbon chains in a HTMA-DS bilayer may be attributed to the strong electrostatic attraction between oppositely charged head groups of HTMA+ and DS−.45 However, the asymmetric hydrocarbon chain lengths of 57
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(D), and hydrogen atom: the acceptor−donor distance (rAD) and the angle formed by the hydrogen, donor, and acceptor atoms (αHDA). When rAD ≤ 0.35 nm and αHDA ≤ 30°, the interaction is defined as a H bond.49,50 Among the bilayer components, the only possible H bond donor is the hydroxyl group of cholesterol, while both hydroxyl groups of cholesterol as well as sulfate group of DS− can be H bond acceptors. The average numbers of H bonds within each bilayer system are given in Table 2. It is revealed that the average numbers of
The results imply that cholesterol can affect the molecular packing in both polar and nonpolar regions of the catanionic bilayers. Because the sterol ring of cholesterol was located near the middle segments of HTMA+ and DS− hydrocarbon chains (Figure 4), the position of sterol ring may be the main factor to affect the molecular packing of the bilayers. The details will be further discussed. 3.3. Bilayer Polar Region. The radial distribution function (RDF), g(r), of specified atom pairs shown in Figure 5 was used
Table 2. H Bonds between DS−, Cholesterol (Chol), and Water for HTMA-DS/Cholesterol Bilayers H bonds XChol
DS-water (no./DS)
Chol-water (no./Chol)
Chol-DS (no./Chol)
Chol-DS (no./membrane)
0 0.09 0.30 0.50
2.56 2.59 3.00 3.55
0.85 1.19 1.70
0.77 0.60 0.43
9.25 22.97 27.42
H bonds with water per DS− as well as those per cholesterol increased with increased XChol. This is indicative of more water molecules surrounding the sulfate group of DS− and hydroxyl groups of cholesterol due to a cholesterol-induced space in the polar region of a bilayer. Furthermore, when XChol was increased, more H bonds were formed between DS− and cholesterol in the bilayers. The resulting H bonds between the head groups of DS− and cholesterol together with the strong hydrophobicity of cholesterol may attract DS− moiety toward the hydrophobic region of the bilayer. This gives an explanation for DS− head groups shifting toward the inside of the bilayer with the addition of cholesterol. 3.4. Bilayer Nonpolar Region. The microscopic structure of bilayer components and the order of hydrocarbon chains are normally characterized by the order parameter tensor, Sαβ, defined as51 1 Sαβ = ⟨3 cos θα cos θβ − δαβ⟩, a , β = x , y , z (2) 2 where θα is the angle between the αth molecular axis and the bilayer normal (z-axis) and δαβ represents the Kronecker delta function. The angle bracket denotes an average for all molecules and time. In this study, the experimentally relevant deuterium order parameter defined as SCD = (2Sxx + Syy)/3 was used to investigate the segment order of the hydrocarbon chains. Order parameter profiles calculated for HTMA+ and DS− in each system are displayed in Figure 6. |SCD| profiles of HTMA+ and DS− hydrocarbon chains in the HTMA-DS bilayer showed a plateau at ∼0.4, except for a significant decrease in the tail segments of HTMA+. This was indicative of a highly ordered structure in the HTMA-DS bilayer, except for the tail segments of HTMA+. Because HTMA+ and DS− possess hydrocarbon chains of C16 and C12, respectively, the additional four carbons in the HTMA+ hydrocarbon chain may cause disordered packing in the tail segments.45 With the presence of cholesterol, the order parameters for HTMA+ and DS− hydrocarbon chains were significantly increased, especially in the middle segments of HTMA+ and DS− hydrocarbon chains. This might be attributed to the insertion of the rigid cholesterol ring, which can restrain neighboring hydrocarbon chain segments from motion.19,52
Figure 5. Radial distribution functions of specified atom pairs: (a) N− S, (b) N−N, and (c) S−S.
to examine the relative distance between the head groups of the bilayer-forming molecules. It is found that the initial peak position of gNS(r) was not significantly changed upon the inclusion of cholesterol. However, with increased XChol, the initial peak heights of gNN(r) and gSS(r) decreased, and the second peak heights increased. This indicates that the addition of cholesterol could increase the distance between head groups of HTMA-DS molecules but was incapable of separating HTMA+ and DS−. It is worthy to note that hydroxyl groups of cholesterol were located at the inside of the bilayer in comparison with the head groups of HTMA+ and DS−. Apparently, the addition of cholesterol can create a space in the polar region of the bilayer, and thus the interaction between water and head groups of the bilayer components may be enhanced. Hydrogen (H) bonds within each bilayer system were also analyzed. Hydrogen bonds were defined by referring to geometric criteria among all possible acceptor (A), donor 58
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content of cholesterol promotes a more order of molecular orientation but lowers the order of molecular conformation. To clarify the effect of cholesterol on molecular packing of the rigid HTMA-DS bilayer, probability profiles of the gauche conformers along the HTMA+ and DS− hydrocarbon chains are plotted in Figure 7. The gauche conformer probabilities of
Figure 6. Order parameter profiles along the hydrocarbon chains of (a) HTMA and (b) DS with different cholesterol contents.
The average values of tilt angles for bilayer-forming molecules are listed in Table 3. The tilt angles of HTMA+ Table 3. Average Values of the Chain Tilt Angles and Gauche Fractions in HTMA-DS/Cholesterol Bilayers tilt angle [deg] XChol
HTMA
DS
0 0.09 0.30 0.50
13.3 11.4 8.6 9.5
12.4 11.5 7.5 8.3
Figure 7. Probabilities of gauche conformers along the hydrocarbon chains of (a) HTMA and (b) DS with different cholesterol contents.
gauche fraction [%] Chol ring
HTMA
DS
13.5 7.6 7.8
8.3 7.2 8.0 9.9
4.0 2.1 2.7 4.6
middle segments of HTMA+ and DS− hydrocarbon chains were decreased with the addition of cholesterol. On the contrary, the gauche conformer probabilities of terminal segments of HTMA+ and DS− hydrocarbon chains together with front segment of HTMA+ hydrocarbon chain were increased by increased XChol. It is noteworthy that the cholesterol ring was located near the middle segments of HTMA+ and DS− hydrocarbon chains (Figure 4). Thus, the reduced gauche conformer probabilities of the middle segments of HTMA+ and DS− hydrocarbon chains were attributed to the inserted rigid sterol ring of cholesterol which restrained the motion of neighboring hydrocarbon chain segments.19,52 In contrast, the pronounced void in the hydrophobic core induced by cholesterol increased the motion freedom of the terminal segments of the hydrocarbon chains, leading to the increment in the gauche conformer probabilities. In the polar region of the bilayers, the presence of cholesterol created a space between the head groups of HTMA-DS as well and induced a shift of DS− head groups toward the inside of the bilayer. Thus, the head groups of HTMA+ protruded out of the bilayer, leading to an increase in the gauche conformer probability of front segments of the HTMA+ hydrocarbon chains. Apparently, the spacing effect, involved in the polar and nonpolar regions of the bilayers, inducing a conformational disorder was more pronounced with increased XChol, and hence the disordering effect may overwhelm the ordering effect induced by the cholesterol ring, resulting in the large average gauche conformer probability in the HTMA-DS bilayers with high XChol. 3.5. Condensing Effect. One of the widely discussed and documented effects of cholesterol is the condensing effect on
and DS− hydrocarbon chains are defined as the angle between the vector connecting the first and the last carbon atoms of the hydrocarbon chains and the bilayer normal. The tilt angle of cholesterol ring is evaluated as the angle between the C3−C17 vector and the bilayer normal. In the HTMA-DS bilayer, the average tilt angle of HTMA+ and DS− hydrocarbon chains was ∼13°. The average tilt angle became small with the addition of cholesterol and reached a value of ∼8° at XChol ≥ 0.3. The tendency of tilt angle change for cholesterol ring was similar to that for HTMA+ and DS− hydrocarbon chains. The average tilt angles of cholesterol ring at high XChol were smaller than that at XChol = 0.09, and the values were also close to 8°. Recently, the sterol tilt has been demonstrated to be the major factor in affecting the ordering capability of sterols in membranes.53,54 That is a smaller tilt angle implies a greater order of the bilayer. Thus, the tilt angle results of the bilayer components indicated that cholesterol ring together with HTMA + and DS − hydrocarbon chains tended parallel with the bilayer normal and formed an ordered bilayer. The average fractions of gauche conformers for whole HTMA+ and DS− hydrocarbon chains in each system are also given in Table 3. The average gauche fractions of HTMA+ and DS− hydrocarbon chains were smallest at XChol = 0.09 and gradually increased with increased XChol. At XChol = 0.5, the average values of gauche fractions were larger than those in the HTMA-DS bilayer. It is an interesting finding that a higher 59
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fluid lipid bilayers,22 which is defined as a decrease of moleculeoccupied area induced by cholesterol.19,22 The condensing effect always accompanies a reduction of gauche defects and ordered molecular orientation,55,56 reflecting the increase of bilayer thickness.22 In this study, cholesterol induced a condensing effect on the rigid bilayer composed of HTMADS (Table 1) without changing the bilayer thickness (N−N distance between the average positions of N atoms in opposite leaflets) (Figure 4), although the molecular orientation became ordered. This observation is obviously different from the observed condensing effect on a fluid bilayer. The additive-induced condensation of molecules has been suggested ascribed to enhanced hydrophobic interaction and/ or reduced electrostatic repulsion.56−58 The insertion of cholesterol ring with strong hydrophobicity in the bilayer could effectively enhance the hydrophobic interaction between molecules and promote the tight packing between cholesterol and neighboring molecules.56 Therefore, the condensing effect of cholesterol on the bilayer has been suggested maximizing the contact between cholesterol ring and the neighboring hydrocarbon chains.59−61 The phenomenon is correspondent with the observation in this study that gauche conformer probability decreased for the middle segments of HTMA-DS hydrocarbon chains. However, the spacing effect of cholesterol involved in the polar and nonpolar regions of the bilayers promoted bending in the front and terminal segments of the hydrocarbon chains. This may be the reason to limit the increase in the bilayer thickness. Furthermore, the disordering effect on the front and terminal segments of the hydrocarbon chains may overwhelm the ordering effect on the middle segments of the hydrocarbon chains with high XChol. This also reflects the different condensing effects for the fluid and rigid bilayers.
Apparently, the cholesterol-induced space in the polar region and void in the nonpolar region of the bilayer led to a conformational disorder. With increased XChol, the conformational disorder in the front and terminal segments of the molecule hydrocarbon chains would overwhelm the ordering effect in the middle segments of the molecule hydrocarbon chains. This may be a reason for the cholesterol-induced apparent disordering effect on the rigid HTMA-DS bilayer. This study examined the role of cholesterol in the molecular packing of catanionic HTMA-DS/cholesterol bilayers on the molecular scale, which will be useful for formulating novel catanionic vesicles.
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ASSOCIATED CONTENT
S Supporting Information *
A table showing the compositions of different HTMA-DS/ cholesterol bilayers examined. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel (+) 886-6-2757575 ext 62671; Fax (+) 886-6-2344496; email [email protected] (C.-H.C.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Science Council of Taiwan through Grants NSC98-2221-E-006-098-MY3 and NSC101-2221-E-006-240 and by the high performance cluster computing service from computer and network center of National Cheng Kung University.
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4. CONCLUSION A series of all-atom molecular dynamics simulations were conducted to investigate the effects of cholesterol on the molecular packing of catanionic bilayer composed of a pseudodouble-chained IPA, HTMA-DS. It was found that the addition of cholesterol resulted in a condensing effect on the rigid HTMA-DS bilayer, which was slightly different from that on a fluid bilayer. The differences, e.g. an unobvious change of bilayer thickness and a more disordered structure, were ascribed to the space in the polar region and the void in the nonpolar region of the bilayer created by cholesterol ring which was located near the middle segments of HTMA+ and DS− hydrocarbon chains. In the polar region of the bilayer, the addition of cholesterol increased the distance between head groups of HTMA-DS and induced a shift of DS− head groups toward the inside of the bilayer. Thus, the head group of HTMA+ protruded out of the bilayer, leading to an increase in the gauche conformer probability of front segments in the HTMA+ hydrocarbon chains. In the nonpolar region of the bilayer, cholesterol ring tended to be parallel with the bilayer normal and increased the order of the bilayer. In addition, the sterol ring of cholesterol restrained the neighboring hydrocarbon chain segments from motion, leading to a less gauche defect in the middle segments of HTMA+ and DS− hydrocarbon chains. However, the cholesterol-induced void in the hydrophobic core of the bilayer increased the motion freedom of the terminal segments of the hydrocarbon chains, resulting in an increment in the gauche conformer probability.
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(12) Kusumi, A.; Tsuda, M.; Akino, T.; Ohnishi, S.; Terayama, Y. Protein-phospholipid-cholesterol interaction in the photolysis of invertebrate rhodopsin. Biochemistry 1983, 22, 1165−1170. (13) Mouritsen, O. G.; Jørgensen, K. Dynamical order and disorder in lipid bilayers. Chem. Phys. Lipids 1994, 73, 3−25. (14) Bittman, R.; Clejan, S.; Lund-Katz, S.; Phillips, M. C. Influence of cholesterol on bilayers of ester- and ether-linked phospholipids Permeability and 13C-nuclear magnetic resonance measurements. Biochim. Biophys. Acta, Biomembr. 1984, 772, 117−126. (15) Subczynski, W. K.; Wisniewska, A.; Yin, J.-J.; Hyde, J. S.; Kusumi, A. Hydrophobic barriers of lipid bilayer membranes formed by reduction of water penetration by alkyl chain unsaturation and cholesterol. Biochemistry 1994, 33, 7670−7681. (16) El-Sayed, M. Y.; Guion, T. A.; Fayer, M. D. Effect of cholesterol on viscoelastic properties of dipalmitoylphosphatidylcholine multibilayers as measured by a laser-induced ultrasonic probe. Biochemistry 1986, 25, 4825−4832. (17) Bloom, M.; Evans, E.; Mouritsen, O. G. Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective. Q. Rev. Biophys. 1991, 24, 293−397. (18) Kawakami, K.; Nishihara, Y.; Hirano, K. Rigidity of lipid membranes detected by capillary electrophoresis. Langmuir 1999, 15, 1893−1895. (19) Yeagle, P. L. Cholesterol and the cell membrane. Biochim. Biophys. Acta, Rev. Biomembr. 1985, 822, 267−287. (20) Ohvo-Rekilä, H.; Ramstedt, B.; Leppimäki, P.; Peter Slotte, J. Cholesterol interactions with phospholipids in membranes. Prog. Lipid Res. 2002, 41, 66−97. (21) Mannock, D. A.; Lewis, R. N. A. H.; McMullen, T. P. W.; McElhaney, R. N. The effect of variations in phospholipid and sterol structure on the nature of lipid−sterol interactions in lipid bilayer model membranes. Chem. Phys. Lipids 2010, 163, 403−448. (22) Róg, T.; Pasenkiewicz-Gierula, M.; Vattulainen, I.; Karttunen, M. Ordering effects of cholesterol and its analogues. Biochim. Biophys. Acta, Biomembr. 2009, 1788, 97−121. (23) Alwarawrah, M.; Dai, J.; Huang, J. A molecular view of the cholesterol condensing effect in DOPC lipid bilayers. J. Phys. Chem. B 2010, 114, 7516−7523. (24) Olsen, B. N.; Schlesinger, P. H.; Baker, N. A. Perturbations of membrane structure by cholesterol and cholesterol derivatives are determined by sterol orientation. J. Am. Chem. Soc. 2009, 131, 4854− 4865. (25) Murtola, T.; Karttunen, M.; Vattulainen, I. Systematic coarse graining from structure using internal states: Application to phospholipid/cholesterol bilayer. J. Chem. Phys. 2009, 131, 055101− 15. (26) Bennett, W. F. D.; MacCallum, J. L.; Hinner, M. J.; Marrink, S. J.; Tieleman, D. P. Molecular view of cholesterol flip-flop and chemical potential in different membrane environments. J. Am. Chem. Soc. 2009, 131, 12714−12720. (27) de Meyer, F.; Smit, B. Effect of cholesterol on the structure of a phospholipid bilayer. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 3654− 3658. (28) Khelashvili, G.; Harries, D. How sterol tilt regulates properties and organization of lipid membranes and membrane insertions. Chem. Phys. Lipids 2013, 169, 113−123. (29) Khelashvili, G.; Harries, D. How cholesterol tilt modulates the mechanical properties of saturated and unsaturated lipid membranes. J. Phys. Chem. B 2013, 117, 2411−2421. (30) Saito, H.; Shinoda, W. Cholesterol effect on water permeability through DPPC and PSM lipid bilayers: a molecular dynamics study. J. Phys. Chem. B 2011, 115, 15241−15250. (31) Chang, W.-F. The effects of additives on the physicochemical properties of cataionic vesicles formed by dihexdecyl-chained ion pair amphiphile. Master Thesis, National Cheng Kung University, Taiwan, 2012. (32) Accelrys Software Inc., Discovery Studio Modeling Environment; Release 3.1. In Accelrys Software Inc., San Diego, 2011. 61
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(55) Smondyrev, A. M.; Berkowitz, M. L. Structure of dipalmitoylphosphatidylcholine/cholesterol bilayer at low and high cholesterol concentrations: molecular dynamics simulation. Biophys. J. 1999, 77, 2075−2089. (56) Róg, T.; Pasenkiewicz-Gierula, M. Cholesterol effects on the phospholipid condensation and packing in the bilayer: a molecular simulation study. FEBS Lett. 2001, 502, 68−71. (57) Sangwai, A. V.; Sureshkumar, R. Coarse-grained molecular dynamics simulations of the sphere to rod transition in surfactant micelles. Langmuir 2011, 27, 6628−6638. (58) Sangwai, A. V.; Sureshkumar, R. Binary interactions and saltinduced coalescence of spherical micelles of cationic surfactants from molecular dynamics simulations. Langmuir 2012, 28, 1127−1135. (59) Sugahara, M.; Uragami, M.; Yan, X.; Regen, S. L. The structural role of cholesterol in biological membranes. J. Am. Chem. Soc. 2001, 123, 7939−7940. (60) Daly, T. A.; Wang, M.; Regen, S. L. The origin of cholesterol’s condensing effect. Langmuir 2011, 27, 2159−2161. (61) Krause, M. R.; Turkyilmaz, S.; Regen, S. L. Surface occupancy plays a major role in cholesterol’s condensing effect. Langmuir 2013, 29, 10303−10306.
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Cholesterol-Induced Condensing and Disordering Effects on a Rigid Catanionic Bilayer: A Molecular Dynamics Study An-Tsung Kuo and Chien-Hsiang Chang* Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan S Supporting Information *
ABSTRACT: Molecular dynamics simulation is applied to investigate the bilayer properties of a novel catanionic vesicle composed of an ion pair amphiphile, hexadecyltrimethylammonium−dodecylsulfate (HTMA-DS), with cholesterol. Structural properties, such as molecular organization, orientation, and conformation, were analyzed from the resulting trajectory. Simulation results showed that cholesterol could induce both condensing and disordering effects on the rigid HTMA-DS bilayer. The condensing effect of cholesterol was ascribed to the maximizing contact between cholesterol ring and the neighboring hydrocarbon chains. Thus, the inserted cholesterol ring restrained the neighboring hydrocarbon chain segments from motion and increased the order of the neighboring hydrocarbon chains. However, the presence of cholesterol would increase the distance between head groups of HTMA-DS and induce a shift of DS− head groups toward the inside of the bilayer. This led to the protrusion of the HTMA+ head groups and conformational disorder in the front segments of HTMA+ hydrocarbon chains. In addition, the cholesterol-induced void in the hydrophobic core of the HTMA-DS bilayer increased the motion freedom of the terminal segments of the hydrocarbon chains. The cholesterol-induced space in the polar region and void in the nonpolar region of the bilayer led to a conformational disorder. With high cholesterol contents, the conformational disorder effect would overwhelm the condensing effect, resulting in the apparent disordering effect on the rigid HTMA-DS bilayer.
1. INTRODUCTION Recently, mixtures of two oppositely charged surfactants, catanionic mixtures or catanionic surfactants, have attracted a lot of attention because of their potential in pharmaceutical applications.1,2 After removing counterions from the mixtures, the complex composed of two oppositely charged amphiphilic moieties is referred as ion pair amphiphile (IPA).2 Both of catanionic surfactant and IPA can form catanionic vesicle, which has the potential as a drug or DNA carrier.1−4 It has been reported that catanionic vesicles prepared from IPAs with a classic mechanical disruption process usually showed poor physical stability.5,6 To improve the physical stability of catanionic vesicles, additives, such as charged surfactants and cholesterol, were introduced in the preparation of vesicles.7−11 It is believed that additional charged surfactants can assist the formation of charged vesicles and cholesterol additive can adjust the molecular packing of the vesicular bilayers. That is the improved intervesicle or intravesicle interaction would promote the formation of stable vesicles. Cholesterol is an important constituent of animal cell membranes. The roles of cholesterol in the cell membranes include regulation of membrane fluidity,12,13 reduction of passive permeability of small molecules, such as water and gases,14,15 and increase of membrane mechanical strength.16−18 The effects of cholesterol on lipid bilayer characteristics have been widely studied.19−22 Cholesterol may also play an © 2013 American Chemical Society
important role in catanionic vesicular bilayer characteristics. The components of catanionic vesicular bilayers are different from lipid bilayers, and the effects of cholesterol on the molecular packing of catanionic vesicular bilayers are not expected the same as those of lipid bilayers. It is thus necessary to explore the effects of cholesterol on the catanionic vesicular bilayer characteristics in order to fully understand the stabilization mechanisms for catanionic vesicles. The molecular dynamics (MD) simulation is a useful tool for investigating the microscopic structures of vesicular bilayers at the molecular level. A large number of MD studies examining the effects of cholesterol on the lipid bilayer characteristics using fully atomistic as well as coarse-grained descriptions have been reported.22−30 These MD studies have provided insights into bilayer properties, such as bilayer structure,22−27 elastic modulus,28,29 and water permeability,30 of lipid bilayers with cholesterol from a molecular viewpoint. With the aim of elucidating how cholesterol affects the molecular packing characteristics of catanionic vesicular bilayers, a series of MD simulations of a catanionic vesicular bilayer composed of IPA with cholesterol were conducted. Because the physical stability of the catanionic vesicle Received: September 23, 2013 Revised: November 16, 2013 Published: December 17, 2013 55
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was adopted for all MD simulations. All simulations were carried out for 50 ns. The period of first 20 ns is considered the equilibration period, and the results obtained in the following 30 ns period were used to analyze the bilayer structure characteristics.
composed of an IPA, hexadecyltrimethylammonium−dodecylsulfate (HTMA-DS), could be enhanced by the addition of cholesterol,31 HTMA-DS was chosen as a model IPA molecule in this work. Structural properties, such as molecular organization, orientation, and conformation, were then analyzed to elucidate the cholesterol effects on molecular packing of catanionic vesicular bilayers.
3. RESULTS AND DISCUSSION 3.1. Average Area per Molecule. The time evolution of the average area per molecule for the HTMA-DS/cholesterol bilayers with various compositions during the last 30 ns is plotted in Figure 2. The average area per molecule significantly
2. SIMULATION METHODS Molecular structure models of hexadecyltrimethylammonium moiety (HTMA+), dodecylsulfate moiety (DS−), and cholesterol were constructed by using Discovery Studio 3.132 (Figure 1). Before constructing the initial structure model of HTMA-
Figure 2. Time evolution of the averaged molecular areas of HTMADS/cholesterol bilayers.
decreased with the increase in the mole fraction of cholesterol (XChol). This reflects the cholesterol-induced condensation of molecular packing within the catanionic bilayers. To confirm the cholesterol-induced condensing effect, the area per HTMADS molecule listed in Table 1 was calculated by subtracting the Table 1. Averaged Molecular Areas (A), Areas per HTMADS (AHTMA‑DS), and Elastic Area Compressibility Moduli (KA) of HTMA-DS/Cholesterol Bilayers Figure 1. Molecular structures of hexadecyltrimethylammonium moiety (HTMA+), dodecylsulfate moiety (DS−), and cholesterol. An ion pair amphiphile (IPA), HTMA-DS, is formed of HTMA+ and DS−.
DS, the HTMA+ and DS− were placed at an appropriately close distance (distance between N and S was approximately 0.4 nm). Next, Packmol33 was used to randomly build the initial structure of the mixed HTMA-DS/cholesterol bilayer. Each bilayer system was composed of 128 surfactant and 3464 water molecules (Table S1 in Supporting Information). The HTMA+, DS−, and cholesterol molecules were parametrized using the CHARMM PARAM27R force field.34,35 The water molecule was described by the TIP3P model.36 All-atom MD simulations were carried out using Gromacs version 4.53 package.37,38 Periodic boundary conditions were applied in all three directions. The MD simulation was performed with an isothermal−isobaric (NPT) ensemble. The temperature of the simulation system was maintained at 298 K by the Nosé−Hoover chain thermostat,39 and the pressure was controlled at 1 bar by the semi-isotropic Parrinello−Rahman barostat.40,41 Long-range electrostatic potential was calculated using the particle-mesh Ewald (PME) method,42 and Lennard-Jones (LJ) pair potentials were evaluated within a cutoff distance of 1.2 nm with a smooth switching function above 1 nm. Constraints were applied to all bonds using the LINCS algorithm.43 The time step size of 2 fs
XChol
A [nm2]
AHTMA‑DS [nm2]
KA [mN/m]
0 0.09 0.30 0.50
0.415 0.392 0.380 0.378
0.415 0.394 0.384 0.386
1967 2412 9551 6305
total cross-section area of cholesterol molecules from the total surface area of the bilayer. With the calculation, the crystalline cholesterol molecular area of ∼0.37 nm2 was used to evaluate the total cross-section area of cholesterol molecules in the bilayer.44 Table 1 apparently demonstrates the condensing effects of cholesterol on the HTMA-DS bilayer; that is, the area per HTMA-DS molecule decreased with increased XChol. Because the HTMA-DS bilayer is in the gel phase at 298 K,45,46 it is thus interesting to note that cholesterol could induce the condensation of hydrocarbon chains in the rigid HTMA-DS bilayer. The details will be discussed in section 3.5. Figure 2 also shows that fluctuations in the average area per molecule attenuated with increased XChol, which may reflect the less flexible bilayer characteristic induced by cholesterol. To compare the mechanical property of the bilayers, the area compressibility modulus, KA, was calculated by using the relationship derived with the linear response theory47,48 KA = 56
kT ⟨A⟩ N ⟨δA2 ⟩
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Figure 3. Snapshots of the HTMA-DS/cholesterol bilayers during MD simulation: (a) HTMA-DS, (b) XChol = 0.09, (c) XChol = 0.30, and (d) XChol = 0.50. (e) The sketch of the cholesterol and neighboring HTMA-DS molecules in the mixed HTMA-DS/cholesterol bilayer. The substances were colored as follows: (blue) HTMA, (red) DS, (yellow) cholesterol, and (cyan) water. H atoms on the surfactants are not shown.
Figure 4. Probability distributions of selected atoms along the bilayer normal: (a) HTMA-DS, (b) XChol = 0.09, (c) XChol = 0.30, and (d) XChol = 0.50.
HTMA+ and DS− could create a void which allowed the long hydrocarbon chains to reorient in the hydrophobic core. This led to a less tight packing near the slip plane. With the inclusion of cholesterol in the HTMA-DS bilayer, regular molecular arrangements of the mixed bilayers were also observed (Figures 3b−d). It appeared that cholesterol molecules tended to be parallel with neighboring hydrocarbon chains (Figure 3e) and located near the bilayer center. Nevertheless, the void in the bilayer center was more pronounced in the presence of cholesterol, and thus the wobble of the terminal segments of the HTMA-DS hydrocarbon chains became significant. The probability distributions of selected atoms along the bilayer normal (z-axis) are plotted in Figure 4. In the HTMADS bilayer, the nitrogen (N) and sulfur (S) atoms in HTMADS head groups located almost at the same z-position. The peak position of N in the HTMA+ head group was not changed with the presence of cholesterol. However, the peak position of S in the DS− head group was slightly shifted toward the inside of the bilayer and tended to locate between the head groups of HTMA+ and cholesterol when XChol was increased.
where A is the averaged cross-sectional area per molecule and N is the number of molecules aligned in a bilayer leaflet. The angle brackets denote the ensemble average. This equation indicates that the bilayer area compressibility modulus is inversely proportional to area fluctuation. The calculated moduli of mixed HTMA-DS/cholesterol bilayers are listed in Table 1. With the incorporation of cholesterol, the KA value of the mixed bilayer became large and achieved the maximum at XChol = 0.3. Because the higher area compressibility modulus of a bilayer generally corresponds to the higher tensile strength,47 it is suggested that the addition of cholesterol can enhance the stability of the bilayers under applied external mechanical forces. 3.2. Overall Bilayer Structure. Figure 3 displays the snapshot of each bilayer system. A snapshot of the HTMA-DS bilayer is shown in panel a. Hydrocarbon chains of a HTMADS bilayer were in a regular arrangement, except for those near the slip plane between the two bilayer leaflets. The regular arrangement of the hydrocarbon chains in a HTMA-DS bilayer may be attributed to the strong electrostatic attraction between oppositely charged head groups of HTMA+ and DS−.45 However, the asymmetric hydrocarbon chain lengths of 57
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(D), and hydrogen atom: the acceptor−donor distance (rAD) and the angle formed by the hydrogen, donor, and acceptor atoms (αHDA). When rAD ≤ 0.35 nm and αHDA ≤ 30°, the interaction is defined as a H bond.49,50 Among the bilayer components, the only possible H bond donor is the hydroxyl group of cholesterol, while both hydroxyl groups of cholesterol as well as sulfate group of DS− can be H bond acceptors. The average numbers of H bonds within each bilayer system are given in Table 2. It is revealed that the average numbers of
The results imply that cholesterol can affect the molecular packing in both polar and nonpolar regions of the catanionic bilayers. Because the sterol ring of cholesterol was located near the middle segments of HTMA+ and DS− hydrocarbon chains (Figure 4), the position of sterol ring may be the main factor to affect the molecular packing of the bilayers. The details will be further discussed. 3.3. Bilayer Polar Region. The radial distribution function (RDF), g(r), of specified atom pairs shown in Figure 5 was used
Table 2. H Bonds between DS−, Cholesterol (Chol), and Water for HTMA-DS/Cholesterol Bilayers H bonds XChol
DS-water (no./DS)
Chol-water (no./Chol)
Chol-DS (no./Chol)
Chol-DS (no./membrane)
0 0.09 0.30 0.50
2.56 2.59 3.00 3.55
0.85 1.19 1.70
0.77 0.60 0.43
9.25 22.97 27.42
H bonds with water per DS− as well as those per cholesterol increased with increased XChol. This is indicative of more water molecules surrounding the sulfate group of DS− and hydroxyl groups of cholesterol due to a cholesterol-induced space in the polar region of a bilayer. Furthermore, when XChol was increased, more H bonds were formed between DS− and cholesterol in the bilayers. The resulting H bonds between the head groups of DS− and cholesterol together with the strong hydrophobicity of cholesterol may attract DS− moiety toward the hydrophobic region of the bilayer. This gives an explanation for DS− head groups shifting toward the inside of the bilayer with the addition of cholesterol. 3.4. Bilayer Nonpolar Region. The microscopic structure of bilayer components and the order of hydrocarbon chains are normally characterized by the order parameter tensor, Sαβ, defined as51 1 Sαβ = ⟨3 cos θα cos θβ − δαβ⟩, a , β = x , y , z (2) 2 where θα is the angle between the αth molecular axis and the bilayer normal (z-axis) and δαβ represents the Kronecker delta function. The angle bracket denotes an average for all molecules and time. In this study, the experimentally relevant deuterium order parameter defined as SCD = (2Sxx + Syy)/3 was used to investigate the segment order of the hydrocarbon chains. Order parameter profiles calculated for HTMA+ and DS− in each system are displayed in Figure 6. |SCD| profiles of HTMA+ and DS− hydrocarbon chains in the HTMA-DS bilayer showed a plateau at ∼0.4, except for a significant decrease in the tail segments of HTMA+. This was indicative of a highly ordered structure in the HTMA-DS bilayer, except for the tail segments of HTMA+. Because HTMA+ and DS− possess hydrocarbon chains of C16 and C12, respectively, the additional four carbons in the HTMA+ hydrocarbon chain may cause disordered packing in the tail segments.45 With the presence of cholesterol, the order parameters for HTMA+ and DS− hydrocarbon chains were significantly increased, especially in the middle segments of HTMA+ and DS− hydrocarbon chains. This might be attributed to the insertion of the rigid cholesterol ring, which can restrain neighboring hydrocarbon chain segments from motion.19,52
Figure 5. Radial distribution functions of specified atom pairs: (a) N− S, (b) N−N, and (c) S−S.
to examine the relative distance between the head groups of the bilayer-forming molecules. It is found that the initial peak position of gNS(r) was not significantly changed upon the inclusion of cholesterol. However, with increased XChol, the initial peak heights of gNN(r) and gSS(r) decreased, and the second peak heights increased. This indicates that the addition of cholesterol could increase the distance between head groups of HTMA-DS molecules but was incapable of separating HTMA+ and DS−. It is worthy to note that hydroxyl groups of cholesterol were located at the inside of the bilayer in comparison with the head groups of HTMA+ and DS−. Apparently, the addition of cholesterol can create a space in the polar region of the bilayer, and thus the interaction between water and head groups of the bilayer components may be enhanced. Hydrogen (H) bonds within each bilayer system were also analyzed. Hydrogen bonds were defined by referring to geometric criteria among all possible acceptor (A), donor 58
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content of cholesterol promotes a more order of molecular orientation but lowers the order of molecular conformation. To clarify the effect of cholesterol on molecular packing of the rigid HTMA-DS bilayer, probability profiles of the gauche conformers along the HTMA+ and DS− hydrocarbon chains are plotted in Figure 7. The gauche conformer probabilities of
Figure 6. Order parameter profiles along the hydrocarbon chains of (a) HTMA and (b) DS with different cholesterol contents.
The average values of tilt angles for bilayer-forming molecules are listed in Table 3. The tilt angles of HTMA+ Table 3. Average Values of the Chain Tilt Angles and Gauche Fractions in HTMA-DS/Cholesterol Bilayers tilt angle [deg] XChol
HTMA
DS
0 0.09 0.30 0.50
13.3 11.4 8.6 9.5
12.4 11.5 7.5 8.3
Figure 7. Probabilities of gauche conformers along the hydrocarbon chains of (a) HTMA and (b) DS with different cholesterol contents.
gauche fraction [%] Chol ring
HTMA
DS
13.5 7.6 7.8
8.3 7.2 8.0 9.9
4.0 2.1 2.7 4.6
middle segments of HTMA+ and DS− hydrocarbon chains were decreased with the addition of cholesterol. On the contrary, the gauche conformer probabilities of terminal segments of HTMA+ and DS− hydrocarbon chains together with front segment of HTMA+ hydrocarbon chain were increased by increased XChol. It is noteworthy that the cholesterol ring was located near the middle segments of HTMA+ and DS− hydrocarbon chains (Figure 4). Thus, the reduced gauche conformer probabilities of the middle segments of HTMA+ and DS− hydrocarbon chains were attributed to the inserted rigid sterol ring of cholesterol which restrained the motion of neighboring hydrocarbon chain segments.19,52 In contrast, the pronounced void in the hydrophobic core induced by cholesterol increased the motion freedom of the terminal segments of the hydrocarbon chains, leading to the increment in the gauche conformer probabilities. In the polar region of the bilayers, the presence of cholesterol created a space between the head groups of HTMA-DS as well and induced a shift of DS− head groups toward the inside of the bilayer. Thus, the head groups of HTMA+ protruded out of the bilayer, leading to an increase in the gauche conformer probability of front segments of the HTMA+ hydrocarbon chains. Apparently, the spacing effect, involved in the polar and nonpolar regions of the bilayers, inducing a conformational disorder was more pronounced with increased XChol, and hence the disordering effect may overwhelm the ordering effect induced by the cholesterol ring, resulting in the large average gauche conformer probability in the HTMA-DS bilayers with high XChol. 3.5. Condensing Effect. One of the widely discussed and documented effects of cholesterol is the condensing effect on
and DS− hydrocarbon chains are defined as the angle between the vector connecting the first and the last carbon atoms of the hydrocarbon chains and the bilayer normal. The tilt angle of cholesterol ring is evaluated as the angle between the C3−C17 vector and the bilayer normal. In the HTMA-DS bilayer, the average tilt angle of HTMA+ and DS− hydrocarbon chains was ∼13°. The average tilt angle became small with the addition of cholesterol and reached a value of ∼8° at XChol ≥ 0.3. The tendency of tilt angle change for cholesterol ring was similar to that for HTMA+ and DS− hydrocarbon chains. The average tilt angles of cholesterol ring at high XChol were smaller than that at XChol = 0.09, and the values were also close to 8°. Recently, the sterol tilt has been demonstrated to be the major factor in affecting the ordering capability of sterols in membranes.53,54 That is a smaller tilt angle implies a greater order of the bilayer. Thus, the tilt angle results of the bilayer components indicated that cholesterol ring together with HTMA + and DS − hydrocarbon chains tended parallel with the bilayer normal and formed an ordered bilayer. The average fractions of gauche conformers for whole HTMA+ and DS− hydrocarbon chains in each system are also given in Table 3. The average gauche fractions of HTMA+ and DS− hydrocarbon chains were smallest at XChol = 0.09 and gradually increased with increased XChol. At XChol = 0.5, the average values of gauche fractions were larger than those in the HTMA-DS bilayer. It is an interesting finding that a higher 59
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fluid lipid bilayers,22 which is defined as a decrease of moleculeoccupied area induced by cholesterol.19,22 The condensing effect always accompanies a reduction of gauche defects and ordered molecular orientation,55,56 reflecting the increase of bilayer thickness.22 In this study, cholesterol induced a condensing effect on the rigid bilayer composed of HTMADS (Table 1) without changing the bilayer thickness (N−N distance between the average positions of N atoms in opposite leaflets) (Figure 4), although the molecular orientation became ordered. This observation is obviously different from the observed condensing effect on a fluid bilayer. The additive-induced condensation of molecules has been suggested ascribed to enhanced hydrophobic interaction and/ or reduced electrostatic repulsion.56−58 The insertion of cholesterol ring with strong hydrophobicity in the bilayer could effectively enhance the hydrophobic interaction between molecules and promote the tight packing between cholesterol and neighboring molecules.56 Therefore, the condensing effect of cholesterol on the bilayer has been suggested maximizing the contact between cholesterol ring and the neighboring hydrocarbon chains.59−61 The phenomenon is correspondent with the observation in this study that gauche conformer probability decreased for the middle segments of HTMA-DS hydrocarbon chains. However, the spacing effect of cholesterol involved in the polar and nonpolar regions of the bilayers promoted bending in the front and terminal segments of the hydrocarbon chains. This may be the reason to limit the increase in the bilayer thickness. Furthermore, the disordering effect on the front and terminal segments of the hydrocarbon chains may overwhelm the ordering effect on the middle segments of the hydrocarbon chains with high XChol. This also reflects the different condensing effects for the fluid and rigid bilayers.
Apparently, the cholesterol-induced space in the polar region and void in the nonpolar region of the bilayer led to a conformational disorder. With increased XChol, the conformational disorder in the front and terminal segments of the molecule hydrocarbon chains would overwhelm the ordering effect in the middle segments of the molecule hydrocarbon chains. This may be a reason for the cholesterol-induced apparent disordering effect on the rigid HTMA-DS bilayer. This study examined the role of cholesterol in the molecular packing of catanionic HTMA-DS/cholesterol bilayers on the molecular scale, which will be useful for formulating novel catanionic vesicles.
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ASSOCIATED CONTENT
S Supporting Information *
A table showing the compositions of different HTMA-DS/ cholesterol bilayers examined. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel (+) 886-6-2757575 ext 62671; Fax (+) 886-6-2344496; email [email protected] (C.-H.C.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Science Council of Taiwan through Grants NSC98-2221-E-006-098-MY3 and NSC101-2221-E-006-240 and by the high performance cluster computing service from computer and network center of National Cheng Kung University.
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4. CONCLUSION A series of all-atom molecular dynamics simulations were conducted to investigate the effects of cholesterol on the molecular packing of catanionic bilayer composed of a pseudodouble-chained IPA, HTMA-DS. It was found that the addition of cholesterol resulted in a condensing effect on the rigid HTMA-DS bilayer, which was slightly different from that on a fluid bilayer. The differences, e.g. an unobvious change of bilayer thickness and a more disordered structure, were ascribed to the space in the polar region and the void in the nonpolar region of the bilayer created by cholesterol ring which was located near the middle segments of HTMA+ and DS− hydrocarbon chains. In the polar region of the bilayer, the addition of cholesterol increased the distance between head groups of HTMA-DS and induced a shift of DS− head groups toward the inside of the bilayer. Thus, the head group of HTMA+ protruded out of the bilayer, leading to an increase in the gauche conformer probability of front segments in the HTMA+ hydrocarbon chains. In the nonpolar region of the bilayer, cholesterol ring tended to be parallel with the bilayer normal and increased the order of the bilayer. In addition, the sterol ring of cholesterol restrained the neighboring hydrocarbon chain segments from motion, leading to a less gauche defect in the middle segments of HTMA+ and DS− hydrocarbon chains. However, the cholesterol-induced void in the hydrophobic core of the bilayer increased the motion freedom of the terminal segments of the hydrocarbon chains, resulting in an increment in the gauche conformer probability.
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