Cooperative dynamics in a model DPPC membrane arise from membrane layer interactions
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The dynamics of model membranes can be highly heterogeneous, especially in more ordered dense phases. To better understand the origins of this heterogeneity, as well as the degree to which monolayer systems mimic the dynamical properties of bilayer membranes, we use molecular simulations to contrast the dynamical behavior of a single-component dipalmitoylphosphatidylcholine (DPPC) lipid monolayer with that of a DPPC bilayer. DPPC is prevalent in both biological monolayers and bilayers, and we utilize the widely studied MARTINI model to describe the molecular interactions. As expected, our simulations confirm that the lateral structure of the monolayer and bilayer is nearly indistinguishable in both low- and high-density phases. Dynamically, the monolayer and bilayer both exhibit a drop in mobility for dense phases, but we find that there are substantial differences in the amplitude of these changes, as well as the nature of molecular displacements for these systems. Specifically, the monolayer exhibits no apparent cooperativity of the dynamics, while the bilayer shows substantial spatial and temporal heterogeneity in the dynamics. Consequently, the dynamical heterogeneity and cooperativity observed in the bilayer membrane case arises in part due to interlayer interactions. We indeed find a substantial interdigitation of the membrane leaflets which appears to impede molecular rearrangement. On the other hand, the monolayer, like the bilayer, does exhibit complex non-Brownian molecular displacements at intermediate time scales. For the monolayer system, the single particle motion can be well characterized by fractional Brownian motion, rather than being a consequence of strong correlations in the molecular motion previously observed in bilayer membranes. The significant differences in the dynamics of dense monolayers and bilayers suggest that care must be taken when making inferences about membrane dynamics on the basis of monolayer studies.
KeywordsMembrane Dynamics Heterogeneity
Lipid structures are one of the most ubiquitous forms of biological soft matter. Given their vital role in biological function, lipid structures, including lipid monolayers and bilayers, have been the subject of intense study for many decades . Lipid bilayers form membranes that surround all cells and many sub-cellular structures and are rich in membrane proteins. Protein mobility, which is closely connected to membrane function, is highly dependent on the dynamics of the surrounding lipid matrix. While the structure and composition of lipid membranes have been studied extensively, our understanding of their dynamics is still developing . Lipid monolayers form about 90% of pulmonary surfactant, a lipoprotein complex that plays a critical role in lung structure and the breathing process . The absence, deficiency, or impairment of surfactant monolayers can lead to a host of medical problems, including infant respiratory distress syndrome (IRDS), which is a common disorder among premature infants. Consequently, there is widespread interest in understanding the general properties of lipid monolayers and bilayers.
Given the structural similarity between lipid monolayers and bilayers, these systems share a number of physical properties. Indeed, both systems exhibit similar structural properties across a wide range of temperatures and pressures , and thus share similar thermodynamic properties . Both the bilayer and the monolayer also undergo a liquid-liquid phase transition from a high-density phase, which features densely packed lipids at high pressures or low temperatures, to a low-density phase, where lipids are more disordered at low pressures or high temperatures. In many cases, the properties of lipid monolayers are used to infer the properties of bilayers, as experiments on lipid monolayers are often easier to carry out than those on bilayers . However, there are also notable differences between the monolayer and bilayer. Such distinctions are not surprising, given their functional differences: monolayers primarily act as a lubricant in the breathing process; bilayers serve as cell membranes and compartmentalize processes within cells.
While the structural and thermodynamic similarities between monolayers and membranes are documented, studies contrasting the dynamics of these lipid systems are comparatively less common. The consensus view on the nature of lipid dynamics has evolved substantially over the past several decades . The fluid mosaic model , in which all elements undergo independent and uncorrelated lateral motion, has been supplanted on the basis of evidence for more complex correlations in lipid rearrangement [8, 9]. In particular, the concept of functional “lipid rafts” that are structurally and dynamically distinct from their surroundings [10, 11] has become a predominant view. It is widely agreed that these rafts are characterized by heterogeneous dynamics within the lipid layer that involves a combination of lipids and the protein Caveolin-1, though the quantitative description of these structures remains a broadly studied and debated topic.
Experiments and simulations on lipid layers have shown that such heterogeneity arises as a fundamental property of lipid systems [12, 13, 14, 15, 16], even in the absence of compositional variations. For example, neutron scattering experiments on single-component bilayers have demonstrated that lipids exhibit short-term localized mobility consistent with the formation of dynamical clusters , and experiments on single-component lipid monolayers observed heterogeneous rotational dynamics . Simulations of pure DPPC lipid bilayers show dynamic heterogeneity in the form of lipid clusters on the size and time scales expected for lipid rafts, solely as a result of the intrinsic dynamics of the membrane lipids . While peptides and proteins play a vital role in the functional heterogeneous dynamics of living membranes, it is apparent that heterogeneity can arise even without the complexity of these multiphase membranes. Accordingly, it is important to understand the underlying framework upon which these complex biomolecules must dance.
Such dynamically heterogeneous behavior is similar to that already well-established in a variety of soft condensed matter systems when the intermolecular interactions are strong relative to the thermal energy, such as occurs in simple fluids approaching a glass transition, including polymers and granular materials [19, 20]. This heterogeneity is characterized by the distinction between mesoscopic regions of varying mobility and frequently occurs without any significant change in the overall structure of these systems.
In this manuscript, we study the dynamics of simulated single-component monolayers and bilayers comprised of dipalmitoylphosphatidylcholine (DPPC) lipids to assess the degree to which the dynamics of monolayer systems mimic those of bilayer membranes, particularly with regard to cooperative lipid rearrangements. We focus on DPPC since it is the most common lipid component of pulmonary surfactant and is also prevalent in cell membranes . DPPC has shown to exhibit cooperative lipid motions in single-component simulations of bilayers [13, 15], and it is one of the most frequently studied lipids . While our single-component lipid structures are a substantial simplification in comparison to the multicomponent monolayers and membranes found in biological systems, these model systems allow us to explore the fundamental dynamic behavior of lipid monolayers and bilayers without the presence of multiple lipid, protein, and other membrane localized molecules (e.g., cholesterol, etc.) that complicate our understanding of the underlying mechanism responsible for dynamical heterogeneity. In our view, studying these “simple” lipid systems is a necessary early step in a bottom-up approach to comprehend the dynamics of real biological membranes; we must crawl a little before we run a marathon.
Our findings are based on molecular dynamics (MD) simulations of single-component DPPC lipid layers modeled by the coarse-grained (CG) MARTINI force field . Our simulations confirm the expected similarities in the thermodynamic and structural properties of membranes and monolayers, but we find significant differences in dynamics of these lipid structures. In particular, the monolayer systems show little evidence for cooperativity of lipid displacements, unlike the behavior of lipids in membranes. Significant interdigitation of the lipid layers in membranes apparently plays an important role in the cooperativity of molecular rearrangements. These findings illustrate the potential challenges of using monolayers as model systems to describe membranes.
2 Model and simulations
We performed molecular dynamics simulations of DPPC monolayers and bilayers using the coarse-grained MARTINI model, which has been systematically parameterized to reproduce thermodynamic properties of lipid membranes . We select DPPC because it is the most common component of surfactant monolayers and is also prevalent in cell membranes.
Our DPPC monolayer systems consist of two parallel lipid monolayers separated by a vacuum regime and a water regime, which mimics the air-water interface where lipid monolayers are found. Periodic boundary conditions are implemented in the x-, y-, and z-directions. Each system includes a total of 2660 lipids, with 1330 lipids per monolayer. There are 66,734 CG water “molecules” separating the monolayers, and, in the MARTINI mapping, each water represents four molecules. Thus, the hydration level far exceeds the minimal amount needed to avoid effects on the dynamics . The DPPC bilayer membrane simulations were reported in our earlier work .
All molecular dynamics simulations were performed using the GROMACS simulation suite. We use a standard integration time step 0.02 ps for the MARTINI model. Temperature and pressure were controlled by the Berendsen algorithm. We implement semi-isotropic pressure coupling, which scales the pressure in the x-y plane independently from that of the z-direction. Pressure coupling in the x-y plane is set to zero and is independent of pressure fluctuations in the z-direction, allowing us to maintain a surface tension equal to zero. Furthermore, the box in the z- direction is fixed so that the vacuum regime remains stable.
We generated initial equilibrium structures by performing 1-μs equilibration runs at temperatures between 300 and 350 K at zero surface tension. Given the propensity for hysteresis in the transition between expanded and condensed states , we equilibrated systems near the phase transition starting from both expanded and condensed states. Following this equilibration, “production” simulations were carried out, from which molecular coordinates were stored, and all data we shall show was collected. For systems in the liquid-expanded phase, these production runs were an additional 1 μs in duration; for the less mobile liquid-condensed phase, production runs were an additional 3 μs in duration.
We now proceed to contrast the dynamical properties of these systems. Given the strong similarity in leaflet structure of the bilayer and monolayer, one might expect similar dynamical behavior. However, we shall show that there are significant differences in the nature of the local lipid rearrangements that we did not anticipate.
This strong sub-diffusive behavior observed in the monolayer and membrane is also a characteristic feature of many glass-forming fluids [20, 36, 37, 38]. The weak dependence of 〈r2(t)〉 on time indicates that lipids remain in a localized region close to their initial positions. This transient molecular trapping is often referred to as molecular “caging.” In glass-forming systems and in our previous simulations on membrane systems [14, 15, 16], molecular caging is often associated with cooperative motion in the form of heterogeneous mesoscopic clusters of molecular units having relatively high and low mobility in comparison to ensembles of Brownian particles having the same average diffusion coefficient , a phenomenon referred to as “dynamical heterogeneity.” We next explore the degree to which such collective molecular motion occurs in the lipid monolayer.
4 Discussion and conclusion
We performed molecular dynamics simulations of lipid systems in order to contrast the dynamical behavior of lipid monolayers and bilayers and provide further insight into the origins of cooperative motions within a model DPPC bilayer membrane. Although our single-component systems are simplifications of actual biological structures, they allow us to better understand the fundamental mechanisms of cooperative lipid motions that are vital to biological function. We confirmed that lipid monolayers and bilayers exhibit very similar thermodynamic and structural organization. In this regard, the lipid monolayer can indeed be considered a helpful model to understand the properties of the bilayer membrane. However, while monolayers and bilayers exhibit similarities in their mean dynamics, such as decreased diffusivity in the high-density phase, the nature of the molecular displacements and their cooperativity is fundamentally different for monolayers and membranes: membranes exhibit spatial and temporal heterogeneity of the lipid displacements in the high-density phase, while monolayers do not. Because lipid structure within the leaflet of lipid monolayers and bilayers is nearly identical, inter-leaflet interactions in the bilayer are the origin for the observed differences between monolayers and bilayers.
A more complete understanding of the dynamics of lipid monolayers and bilayers informs experimental approaches as well as the conceptual framework upon which models of lipid dynamics are built. Our comparative study of these two lipid systems suggests that inter-leaflet interactions in membranes play an important role in the emergence of dynamical heterogeneity and, possibly, the formation of lipid rafts. Though again, we must emphasize that functional rafts depend on the interactions among the lipids, peptides, and proteins in the membrane; indeed, antimicrobial peptides such as Alamethicin can potentially disrupt raft formation [43, 44]. Experiments on lipid monolayers, which are typically easier to carry out than those on bilayers, are often used to infer properties of cell membranes; our study of the structure and dynamic behavior of these systems informs on both the strengths and limitations of this application. The substantial similarities between monolayers and bilayers in the low-density phase suggest that monolayers may be used as a model system to understand bilayers. In contrast, the dynamical behavior of high-density monolayers is qualitatively different than that of the corresponding bilayers, at least in the case of our simulated DPPC.
The next step in a comprehensive, bottom-up, approach to understanding lipid dynamics is to include other lipid types in our systems. Cholesterol is the natural choice as a second lipid component because of its prevalence in lipid monolayers and bilayers and its association with lipid raft formation in the literature . Comparative studies of lipid bilayers and monolayers might reveal cholesterol’s underlying role in the formation of lipid rafts. We can compare DPPC-cholesterol systems to our single-component DPPC “baseline” systems to better understand how cholesterol affects lipid clustering and heterogeneous dynamics; we can compare DPPC-cholesterol monolayers and bilayers to better understand how cholesterol flip-flopping, which leads to increased inter-leaflet interaction, might affect bilayer dynamics. Further considerations include the study of lipid monolayers at different pressures, relevant to pressure changes in surfactant monolayers.
We thank C. Othon and I. Mukerji for discussions. Computer time was provided by Wesleyan University. This work was supported in part by NIST award 70NANB15H282.
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