Langmuir 2007, 23, 12465-12468
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Molecular-Scale Structure in Fluid-Gel Patterned Bilayers: Stability of Interfaces and Transmembrane Distribution Sandra V. Bennun, Marjorie L. Longo, and Roland Faller* Department of Chemical Engineering and Materials Science, UniVersity of CaliforniasDaVis, DaVis, California 95616 ReceiVed May 11, 2007. In Final Form: October 6, 2007 Variations in two-dimensional membrane structures on the molecular length scale are considered to have an effect on the mechanisms by which living cell membranes maintain their functionality. We created a molecular model of a patterned bilayer to asses the static and dynamic variations of membrane lateral and transbilayer distribution in two-component lipid bilayers on the molecular level. We study DSPC (distearoylphosphatidylcholine) nanometer domains in a fluid DLPC (dilauroylphosphatidylcholine) background. The system exhibits coexisting fluid and gel phases and is studied on a microsecond time scale. We characterize three different kinds of patterns: symmetric domains, asymmetric domains, and symmetric-asymmetric domains. Preferred bilayer configurations on the nanoscale are those that minimize the hydrophobic mismatch. We find nanoscale patterns to be dynamic structures with mainly lateral and rotational diffusion affecting their stability on the microsecond time scale.
Although biological membranes are characterized by their fluidity, it is hypothesized that two-dimensional membrane heterogeneities, resulting from changes in the membrane lateral and transbilayer distribution,1,2 play a fundamental role in controlling membrane functionality. The mechanisms that control the dynamic structure of cell membranes involve elaborate and complex lipid and protein interactions. Considerable research effort has focused on characterizing lipid organization in simplified model membrane systems as a means of inferring the complex interactions of lipids within real cell membranes. The characterization of coexisting lipid phases in model systems such as large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs),3-5 small unilamellar vesicles (SUVs),6 supported lipid bilayers (SLBs),7 and lipid monolayers2 has provided a great deal of information, often on the micrometer scale. Studies on GUVs suggest that gel domains ∼30 µm in size extend from the inner to the outer leaflet.5 Transmembrane segregation was found in SUVs,6 and detailed AFM studies on SLBs7 show both symmetric and asymmetric domains and indicate that the leaflet compositions depends very much on the type of lipids in the mixture and their thermal history. Nearest-neighbor recognition (NNR)3 on LUVs allows to recognize the presence of domains for lipid mixtures differing by four methylene groups in the fluid-gel phase coexistence region, but information about transbilayer complementarity was obtained only in the pure liquidcrystalline phase.4 Compositional variation within gel domains has been observed recently, at 100 nm resolution, using secondary ion mass spectrometry.8 However, the details of membrane organization on scales below 100 nm have mainly been the realm of computer simulation.9-13 Two component fluid- and gel-phase coexistence bilayers have been rarely addresses for large time * Corresponding author. E-mail:
[email protected]. (1) Brown, D. A.; London, E. J. Biol. Chem. 2000, 275, 17221-17224. (2) Gennis, R. B. Biomembranes: Molecular Structure and Function; SpringerVerlag: New York, 1989. (3) Krisovitch, S. M.; Regen, S. L. J. Am. Chem. Soc. 1992, 114, 9828-9835. (4) Zhang, J. B.; Jing, B. W.; Tokutake, N.; Regen, S. L. J. Am. Chem. Soc. 2004, 126, 10856-10857. (5) Bagatolli, L. A.; Gratton, E. Biophys. J. 2000, 79, 434-447. (6) Op den Kamp, J. A. F. Annu. ReV. Biochem. 1979, 48, 47-71. (7) Lin, W. C.; Blanchette, C. D.; Ratto, T. V.; Longo, M. L. Biophys. J. 2006, 90, 228-237. (8) Kraft, M. L.; Weber, P. K.; Longo, M. L.; Hutcheon, I. D.; Boxer, S. G. Science 2006, 313, 1948-1951. (9) Marrink, S. J.; Mark, A. E. Biophys. J. 2004, 87, 3894-3900.
scales.14 Atomistic simulations showed that asymmetry in the membrane induces a nonzero membrane potential in the absence of ions,15 suggesting that lipid asymmetry, a characteristics of living cells, can contribute to the membrane potential. Coarsegrained simulations of single-component lipid membranes addressed in detail the stages of the transformation between the gel and the fluid phase and showed that the nucleation of a stable gel cluster of ∼20 to 80 lipids encompassing both leaflets is the critical step in gel-phase formation.10 To infer mechanisms by which cell membranes maintain their lateral and transmenbrane distribution reconciliation of data on different time and length scales is required, as is a comparison of data under similar conditions in the same system, ideally on the same length scale. In this letter, we address lipid-lipid interactions and lateral and transbilayer organization within a fluid-gel coexisting lipid membrane on length scales of tens of nanometers. Recently, we studied the phase and mixing behavior of DSPC-DLPC phospholipid bilayers by mesoscale molecular modeling11,16,17 and composition as well and lateral and transbilayer reorganization of this system by AFM experiments on larger length scales.7,8,18 We perform molecular dynamics simulations of the DSPCDLPC mixture and focus on understanding the lateral and transbilayer pattern reorganization and stability of nanodomains using a molecular model. The initial simulation conditions mimic experimental patterns observed by our group7 employing AFM and fluorescence microscopy techniques. For that, we created a model of a patterned bilayer using a very well tested coarsegrained lipid model.19,20 Our model system consists of two(10) Marrink, S. J.; Risselada, J.; Mark, A. E. Chem. Phys. Lipids 2005, 135, 223-244. (11) Faller, R.; Marrink, S.-J. Langmuir 2004, 20, 7686-7693. (12) Pandit, S. A.; Jakobsson, E.; Scott, H. L. Biophys. J. 2004, 87, 33123322. (13) Ayton, G. S.; McWhirter, J. L.; McMurty, P.; Voth, G. A. Biophys. J. 2005, 88, 3855-3869. (14) Stevens, M. J. J. Am. Chem. Soc. 2005, 127, 15330-15331. (15) Gurtovenko, A. A.; Vattulainen, I. J. Am. Chem. Soc. 2007, 129, 53585359. (16) Switzer, J.; Bennun, S.; Longo, M. L.; Palazoglu, A.; Faller, R. J. Chem. Phys. 2006, 124, 234906. (17) Bennun, S. V.; Longo, M. L.; Faller, R. J. Phys. Chem. B 2007, 111, 9504-9512. (18) Ratto, T. V.; Longo, M. L. Biophys. J. 2002, 83, 3380-3392. (19) Marrink, S. J.; Vries, A. H. d.; Mark, A. E. J. Phys. Chem. B 2004, 108, 750-760.
10.1021/la701370t CCC: $37.00 © 2007 American Chemical Society Published on Web 11/02/2007
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Figure 2. Two-dimensional radial distribution functions of the C1 centers in the lipid tails for symmetric-asymmetric DSPC domains, where l indicates a single leaflet and b indicates a bilayer. The relative preference of lipid types opposing the same or different lipid types in the bilayer is depicted. The inset shows the DLPC molecule in red and the DSPC molecule in blue.
Figure 1. Snapshots of patterned bilayers of DSPC (blue) and DLPC (red). The left snapshots represent the beginning of the simulation, and the right snapshots represent the final frames of (a-c) 4, (d) 6.7, and (e) 16 µs simulations. Table 1. System Types, Compositions, System Sizes, Simulation Times, and Temperatures system
composition DLPC DSPC
symmetric/asymmetric 1056 656 domain (stripes) 1056 656 528 328 528 328 2128 328 asymmetric 1728 128 1728 128 1728 128 asymmetric (stripes) 528 128 528 128 528 128 528 128 symmetric domains 1600 200 1600 200 1600 200 1600 200 1600 200 symmetric domains 300 200 (stripes) 600 400
system sizes X-Y-Z (nm) 54-8-13.7 48-8.5-11.9 26-8-13 31-8-12.2 29-20-15 19.2-28.5-11 18-24-13.6 19.4-26.7-11.5 22-8.7-10.5 16.3-9.4-12.8 19.8-9.5-10.7 16.4-9.3-12.8 24.9-21.4-12 15-26-13 17.6-28-12 17-25-12 17-25-12.3 17-7.58-12.4 34.3-7.6-12.4
simulation temp time (µs) (K) 4 4 4 4 0.5 2 1.8 4 4 3 4 4 2.6 3.2 4 2.5 4 16.6 6.7
285 280 285 295 295 295 275 280 295 275 285 280 295 275 280 285 280 285 285
component free-standing lipid bilayers of DLPC and DSPC, where fluid-gel patterning can be controlled by temperature, composition, and lipid distribution within both leaflets. The systems are initiated (Figure 1, left) with the transbilayer organizations observed in experiments.7 Patterned bilayers were constructed from bilayers containing pure DLPC in the liquid-crystalline phase or pure DSPC in the gel phase as building blocks. We also use a bilayer with one leaflet of fluid DLPC facing a leaflet of gel DSPC. The assembly of these building blocks resulted in the tree-type patterns depicted in Figure 1 and Table 1. Bilayers were trimmed to ensure the correct size. The systems were energy minimized before an MD run of a few nanoseconds was started (20) Marrink, S. J.; Risselada, H. J.; Yefimov, S.; Tieleman, D. P.; de Vries, A. H. J. Phys. Chem. B 2007, 111, 7812-7824.
with the lipids in restrained positions to allow water equilibration. Finally, regular simulation runs were performed, where the centerof-mass motion of each leaflet was removed at every time step. DLPC and DSPC consist of 10 and 14 interaction sites, respectively (inset of Figure 2). One water bead represents four real waters. The interaction parameters are taken from ref 19. All beads interact through short-range shift-corrected Lennard-Jones potentials; in addition, charged beads also interact via an electrostatic Coulombic potential, which is shifted from 0 to 1.2 nm. The same cutoff distance as for the LJ potential is used. The GROMACS simulation suite21 (version 3.2) has been used to perform all simulations. Time scales have been renormalized by a factor of 4;19 the reported simulation times are real times. We use a time step of 160 fs and keep the temperature and pressure constant using the weak-coupling method.22 For pressure, the coupling is applied to the three Cartesian directions independently with a target value of 1.0 bar. We observed that the lengths of the bilayer in the X, Y, and Z directions typically stabilized between 800 and 1200 ps (Supporting Information). Table 1 gives an overview of our bilayers. They contain between 656 and ∼2500 lipids and are fully hydrated with 14 to 20 CG waters per lipid. Different domain sizes are realized by different numbers of DSPC lipids in the domains. Several independent runs at different temperatures were performed for each system to define the temperature at which a specific pattern will maintain the fluid and gel phases.17 The coexistence state depends on the temperature, system size, pattern type, and thermal history of the patches that compose the patterned bilayer. Simulations at low temperatures yield a gel-phase two-component bilayer, and at 295 K or above there is mixing of the two lipid components. Most of the patterns discussed here show stable fluid- and gelphase coexistence at 285 K and some at 280 K. We differentiate between domains that are completely surrounded in X and Y by a fluid matrix of DLPC from domains that are surrounded by the fluid DLPC matrix only in the X direction (i.e., stripe pattern, Table 1), although results in both cases were similar. This study is limited to the microsecond time scale; to include larger time scale motion types such as flip flop, longer simulations would be required. The fluid-gel coexisting bilayers were simulated for 4 µs unless stated otherwise in Table 1 and for three domain pattern types in Figure 1: (a, b) symmetric-asymmetric domains containing both transbilayer coupled and uncoupled DSPC, (c) asymmetric (uncoupled) DSPC domains, and (d, e), symmetric (21) Van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. C. J. Comput. Chem. 2005, 26, 1701-1718. (22) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684-3690.
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(coupled) DSPC domains. Note that the corresponding temperatures in Figure 1 are about 20-30 K different from that in corresponding experiments as a result of the nature of the coarsegrained model.11,17,19 We also compared for each symmetricasymmetric domain the effect of different (Figure 1a) or identical (Figure 1b) DLPC and DSPC lipid compositions in both leaflets at 285 K because bilayers with different compositions in both leaflets are necessarily under tension. Comparisons among structures are based on evaluations of order parameters of the C2 centers (second tail bead from the head) of the lipid tails, two-dimensional diffusion, radial distribution functions, density profiles, and rotational correlation functions. From comparing Figure 1b-d, we found that the symmetricasymmetric domain (b) possesses higher-order parameters than in parts c and d (the order parameter for coupled and uncoupled DSPC in part b is 0.48), with the coupled part of the DSPC domain still remaining in registry after 4 µs and less diffusion of DSPCs at the domain edges. DLPC facing the ordered DSPC domain across leaflets is highly ordered itself, with respect to both the order parameter and the long-range packing arrangement. The DLPC that is in registry with DLPC in the opposing leaflet is in the liquid-crystalline phase. The 2D radial distribution function (RDF, Figure 2) shows the relative order and arrangement of DLPC and DSPC located in registry within the bilayer and opposing within each leaflet. These radial distribution functions were calculated by maintaining the initial designation of DLPC and DSPC in the leaflet and in the bilayer. However, lipids that during the 4 µs simulation originally were coupled and became uncoupled and vice versa may not keep their initial designation. Nevertheless, calculations of RDFs for several subintervals of the trajectory show consistent results. In the asymmetric domain at 280 K (Figure 1c), DSPC has a smaller order parameter of 0.41 and a larger lateral diffusion of DSPC in fluid DLPC than Figure 1b,d. The DLPC opposing DSPC is less ordered than in the symmetric-asymmetric case (order parameter 0.31), and its fluidity is comparable to that of a pure DLPC bilayer (order parameter 0.31). Lipid flip-flop is not observed on this time scale in agreement with experiments.23 For the symmetric (coupled) domains (Figure 1d,e), the final configurations yield symmetric-asymmetric domains with DSPC clustered in a gel phase (order parameter 0.46), surrounded by fluid DLPC (order parameter 0.32). These order parameters were calculated over the whole trajectory keeping the initial designation of coupled DSPC and DLPC. The simulation of the domain in Figure 1e was run for 16 µs. A cluster analysis was performed in both leaflets (Figure 3) to identify lipids in the gel or fluid phase and lattice defects by computing lipid neighbors through Voronoi analysis10,24 on the coordinates of the C1 beads of the tails. A lipid tail is defined to be in the gel phase if the C1 site has six neighbors with at least five of the neighbors within the first minimum of the radial distribution function for the C1 sites; the distinction of lipids belonging to a specific gel cluster is obtained by defining the maximum of the first neighbor pick of the radial distribution function as the maximum distance between two C1 sites to belong to the same cluster, and a cluster algorithm is employed to connect lipids to gel clusters.10 In our simulation time, we did not observe complete separation of the gel domain in opposing leaflets; however, we see that the gel domain in both leaflets is composed of group dynamics of gel clusters without defects. In Figure 3, we follow the size of the maximum gel cluster in each leaflet over a 16 µs simulation for the bilayer (23) McConnel, H. M.; Kornberg, R. D. Biochemistry 1971, 10, 1111-1120. (24) Shewchuk, J. R. Comput. Geom. 2002, 22, 21-74.
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Figure 3. Maximum cluster size in both leaflets followed over the course of a 16 µs simulation for the symmetric domain bilayer in Figure 1e.
from Figure 1e. The maximum cluster size changes over time as an indicator of domain dynamics, and we do not find leaflet coupling for the maximum cluster at each frame. A simulation run of 2 µs was performed for a configuration of two asymmetric gel domains of DSPC that initially were not in contact and were located in both the upper and lower leaflets. We find that the gel domains preserve their initial uncoupled configuration. To bring domains completely into or out of registry, larger simulation times would be required. Structures presented in Figure 1 reflect that the variety of configurations for this particular system is probably due to the strong hydrophobic mismatch (i.e., the height difference between gel DSPC and fluid DLPC), which we find to be in agreement with experimental values.7 Symmetric DSPC domains are ∼1.7 nm higher than the neighboring fluid phase, and uncoupled DSPC domains are ∼0.95 nm. We noticed partial interdigitation in the opposing DLPC-DSPC leaflets in the case of asymmetric domains, reducing the height difference. A close examination of the domain edge reveals that the line tension in these patterned bilayers (Figure 1) is reduced by the reorganization of the lipids in the fluid-gel interfaces to make a smooth transition between the gel and fluid phases (i.e., to reduce the hydrophobic mismatch). DSPC lipids at the interface are less ordered and adopt a curved shape. Some of them diffuse laterally and intermix with the DLPCs surrounding the domains, resulting in an increase in the concentration of DLPCs at the edges. However, on the time scale of the simulations, complete mixing of the two lipid components is not observed. We tested the reverse case,17 where the starting configurations are completely mixed bilayers of DLPC and DSPC and the final bilayers result in spontaneous mesoscopic phase separation. Figure 4 shows the rotational motion in a lipid bilayer with fluid and gel phase coexistence, and this rotational correlation function was calculated for the C1 centers of the lipid tails for DLPC and DSPC corresponding to the asymmetric-symmetric bilayer in Figure 1b. For this pattern, four populations are identified as DLPCb and DSPCb for lipids coupled to the same type across leaflets and DLPCl and DSPCl for lipids of one type opposing lipids of the other types (asymmetric configuration). It is evident that coupled DSPCb and uncoupled DSPCl present similarly slow rotational motion characteristic of a gel phase whereas coupled DLPCb presents fast rotational motion indicating a liquid-crystalline phase and uncoupled DLPCl presents intermediate motion between a complete gel phase and a liquidcrystalline phase. Because lipid hopping is not observed, rotational
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Figure 4. Rotational correlation function for the C1 centers in the lipids tails for symmetric-asymmetric DSPC domains at 285 K, where l indicates a single leaflet and b indicates a bilayer. The reorientation functions in red are slow and in blue are fast.
and lateral diffusion are the dominant mechanisms of motion on these time scales. In Supporting Information, the lateral diffusion is discussed further. In conclusion, we find that for nanometer domain size in freestanding liquid-gel coexisting bilayers on the microsecond time scale the preferred bilayer configurations are those that minimize the hydrophobic mismatch. In some cases, this may contrast with the expectations of symmetric DSPC domains in GUVs. Interestingly, for asymmetric-symmetric domains, we found slightly more stable domains when both leaflets have identical compositions. This may have an effect on the dynamics of transmembrane distribution in biological membranes and GUVs. We hypothesize that the large hydrophobic mismatch for the coupled DSPC domain may induce transbilayer complementarity at the edges to reduce large line tension due to the small domain size. We observe a variation of the maximum gel cluster size over time for both leaflets. It is possible that DSPC symmetric nanometer-scale domains are not favored because of the high
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hydrophobic mismatch and large perimeter where DSPC and DLPC are in contact. The several nanometer domains studied here, in comparison to micrometer domains, have a much larger perimeter-to-area ratio. Indeed, the symmetric domains in experiments are still larger than those considered in this study. This may indicate that the mechanisms of biomembrane patterning are system composition-, size-, time scale-, and thermal historydependent. We propose that nanoscale patterns in two-component unilamellar free-standing bilayers are dynamic. This suggests that for microsecond time ranges, fluid DLPC and gel DSPC patterned bilayers find the most stable state by undergoing rotational and lateral diffusion. On larger time scales, it is known that lipid flip-flop plays a fundamental role in the mechanism of transmembrane distribution. Acknowledgment. We thank Professor Siewert J. Marrink and Jelger Risselada from the University of Groningen for providing their cluster analysis algorithm. S.V.B. also thanks Chenyue Xing and Allison Dickey for fruitful conversations. This project was supported by the Graduate Research and Education in Adaptive bio-Technology (GREAT) Training Program of the UC systemwide Biotechnology Research and Education Program (grant 2005-244). We also acknowledge Teragrid and the San Diego Supercomputer Center (SDSC) for computer time (projects MCB050018N and TG-MCB050071T). The NSF MRSEC Center for Polymer Interfaces and Macromolecular Assemblies and the NSF NIRT Program (grant CBET 0506662) supported this work as well. Supporting Information Available: Mean-square displacement for the symmetric-asymmetric domains at 285 K corresponding to Figure 1b. Top view of a nanometer-sized coupled DSPC domain surrounded by a fluid matrix of DLPC. Two-dimensional radial distribution functions of the C1 centers in the lipid tails. Longitudinal dimensions in the X, Y, and Z directions for the symmetric domain bilayer in Figure 1f. This material is available free of charge via the Internet at http://pubs.acs.org. LA701370T
