Some Like It Hot: The Effect of Sterols and Hopanoids on Lipid

Letter pubs.acs.org/JPCL

Some Like It Hot: The Effect of Sterols and Hopanoids on Lipid Ordering at High Temperature Bertrand Caron,† Alan E. Mark,†,‡ and David Poger*,† †

School of Chemistry and Molecular Biosciences and ‡Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia S Supporting Information *

ABSTRACT: Sterols and hopanoids have been suggested to reinforce membranes and protect against unfavorable environmental conditions. In particular, hopanoids are found in high concentrations in membranes of thermotolerant and thermophilic bacteria. However, the mechanism whereby sterols and hopanoids stabilize membranes at elevated temperatures is poorly understood. Here, the effect of temperature on the ordering of lipids in bilayers containing cholesterol or the hopanoids bacteriohopanetetrol and diplopterol was explored using molecular dynamics simulations. It is shown that cholesterol induces a high level of ordering over a wide range of temperatures. Bacteriohopanetetrol promotes order within the lipid tails but enhances fluid-like properties of the head groups at high temperatures. In contrast, diplopterol partitions in the midplane of the bilayer. This suggests that individual hopanoids fulfill distinct functions in membranes, with the ordering properties of bacteriohopanetetrol being particularly well suited to maintain the integrity of membranes at temperatures preferred by thermotolerant and thermophilic bacteria. SECTION: Biomaterials, Surfactants, and Membranes

T

he plasma membrane not only acts as a physical barrier that divides and protects the cell from its environment but also fulfills a plethora of other functions, including providing a suitable lipid milieu to membrane proteins and allowing transmembrane transport of molecules. Maintaining both the integrity as well as the dynamic properties of the plasma membrane is essential for cell survival. Variations in the pH, pressure, and temperature can dramatically alter the collective properties of lipid assemblies, including the most stable phase.1−3 In particular, an increase in temperature can cause phosphatidylcholines, phosphatidylethanolamines, and cardiolipins, three types of phospholipids commonly found in eukaryotic and bacterial membranes, to convert from a bilayer-forming lamellar phase to a non-bilayer-forming hexagonal phase.1,4−6 The composition of the membranes of thermotolerant and thermophilic organisms has evolved so as to maintain a fluid lamellar phase. This so-called homeophasic thermal adaptation7 is achieved by modulating the nature of the head groups as well as the acyl chains (length, degree of unsaturation, and branching) 8−10 and by incorporating molecules referred to as membrane reinforcers that stabilize a lamellar structure, such as sterols in eukaryotes and carotenoids and hopanoids in bacteria.11,12 Sterols, such as cholesterol (Figure 1A), have been shown to enhance tolerance to heat shocks in eukaryotes.13−15 They induce the formation of a liquid-ordered phase that combines short-range order of the acyl chains with relative long-range translational disorder.16 At low temperatures, sterols prevent the formation of an ordered gel phase by disrupting the tight packing of the acyl chains, and © 2014 American Chemical Society

Figure 1. Chemical structure of cholesterol, bacteriohopanetetrol, and diplopterol.

at high temperatures, they preserve the lamellar structure of membranes, mitigating the increase of lipid chain disorder due to thermal motions.12,17 Received: September 30, 2014 Accepted: October 17, 2014 Published: October 17, 2014 3953

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Figure 2. Ordering of the POPC molecules in the simulations of pure POPC, POPC/cholesterol, POPC/BHT, and POPC/diplopterol bilayers at 298, 323, 343, and 363 K. (A) Average carbon−hydrogen order parameter ⟨|SCH|⟩ of the sn-1 palmitoyl chain as a function of the simulation temperature. (B) Average mobility uP of the phosphorus atoms along the bilayer normal as a function of the simulation temperature. (C) Snapshots of the POPC/cholesterol and POPC/BHT bilayers at the end of the simulations at 363 K (at 500 and 450 ns, respectively). (D) Snapshots of the POPC/diplopterol bilayer at the end of the simulations at 298, 323, 343, and 363 K. The phosphorus atom and the acyl chains in POPC are depicted as an orange sphere and gray sticks, respectively. Cholesterol, BHT, and diploptene are shown in magenta, blue, and green, respectively.

In this Letter, we examine the influence of cholesterol and the hopanoids BHT and diplopterol on the ordering of lipids in a model bilayer using molecular dynamics simulation at different temperatures, 298, 323, 343, and 363 K. The bilayer consisted of an equimolar mixture of 288 POPC (2-oleoyl-1palmitoyl-sn-glycero-3-phosphocholine) molecules and 288 molecules of cholesterol, BHT, or diplopterol. A 288-POPC bilayer was also simulated at all temperatures for comparison. Details regarding the simulation protocol and the parametrization of hopanoids are available as Supporting Information and in a previous publication.27 To characterize the effect of temperature and lipid composition on lipid chain order, the average order parameter ⟨|SCH|⟩ of the sn-1 palmitoyl chain of POPC was calculated over the last 200 ns of the simulations. The order parameter SCH of a carbon−hydrogen bond measures the relative orientation of the C−H bond with respect to the bilayer normal (taken as the zaxis). SCH can be calculated using

Hopanoids are bacterial pentacyclic triterpenoids based on a hopane skeleton.18 Two common hopanoids are displayed in Figure 1: bacteriohopanetetrol ((32R,33S,34S)-bacteriohopane32,33,34,35-tetrol) and diplopterol (hopan-22-ol). Bacteriohopanetetrol (BHT) is widespread across almost all hopanoidproducing bacteria.19−21 Diplopterol was shown to support the growth of the bacterium Mycoplasma mycoides that normally requires the incorporation of sterols in its plasma membrane.22 Given their apparent structural similarity to sterols, hopanoids have long been considered bacterial surrogates of sterols.23 However, few studies have examined the properties of hopanoids in detail, and little is known about their effect on the structure and dynamics of membranes.19,24−26 Unlike sterols, hopanoids exhibit a great deal of diversity in the size and chemical nature of the substituents attached to the fused ring system. We showed recently that the two hopanoids BHT and diploptene (hop-22(29)-ene) exhibited different behaviors in membranes.27 Specifically, BHT adopted a sterol-like upright orientation in a lipid bilayer, whereas diploptene partitioned in between the leaflets of the bilayer. Furthermore, the condensing and ordering effect of BHT on lipids was found to be weaker than that of cholesterol. Although hopanoids may contribute to enhance order of lipids in membranes in a sterol-like fashion, most likely they have a range of functions in bacterial membranes. In particular, hopanoids have been suggested to stabilize cell membranes at high temperatures.18 O35-Glycosyl and cyclitol ether BHT derivatives have been isolated in thermotolerant and thermophilic bacteria.21,28−30 The thermoacidophilic bacterium Alicyclobacillus acidocaldarius (formerly known as Bacillus acidocaldarius) grows optimally at 45−70 °C (318−343 K).31 In Alicyclobacillus acidocaldarius, BHT derivatives account for 20% of the total lipid content and about a third of all glycolipids.28 Nonetheless, the mechanism whereby BHT and related hopanoids protect membranes in such heat-tolerant bacteria has remained unexplored.

SCH =

1 ⟨3 cos2 β − 1⟩ 2

where β is the angle between a C−H bond and the normal to the bilayer. The angular brackets denote an ensemble average over all of the lipids and the simulation. ⟨|SCH|⟩ was derived from the values of |SCH| for carbons 2−15 in the sn-1 palmitoyl chain of POPC. As the force field used (GROMOS 54A7) is a united-atom force field wherein aliphatic hydrogens are treated implicitly and incorporated into the carbon to which they are bound, the position of the hydrogen was constructed on the basis of the positions of the neighboring carbons atoms assuming sp3 geometry. The variation of ⟨|SCH|⟩ is illustrated in Figure 2A. As can be seen, an increase in temperature led to a clear decrease of ⟨|SCH|⟩ for all of the bilayers studied. In the case of the pure POPC bilayer and the POPC/cholesterol and POPC/BHT bilayers, the change of ⟨|SCH|⟩ was nearly linear 3954

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Figure 3. Variation of the orientational order parameter Θ of the sn-1 palmitoyl chains in POPC as a function of the distance between POPC molecules in the simulations of (A) a POPC bilayer, (B) a POPC/cholesterol bilayer, (C) a POPC/BHT bilayer, and (D) a POPC/diplopterol bilayer at 298, 323, 343, and 363 K.

cholesterol, uP was much smaller at all temperatures (1.7−2.3 nm), indicating a high degree of ordering of the phosphorus atoms. Interestingly, in the POPC/BHT bilayer, uP increased almost linearly from a value close to that in a POPC/ cholesterol at 298 K to a value equivalent to that of a pure POPC bilayer at 363 K. A similar trend was observed for the average width of the peak of the choline head group (see Supporting Information, Figure S2). This would suggest that any role that BHT plays in rigidifying membranes vanishes once the membrane adopts a fluid-like (liquid-disordered) phase. In fact, BHT appears to play a dual role; at temperatures required for the growth of thermotolerant and thermophilic bacteria such as Alicyclobacillus acidocaldarius, it induces stiffening of the acyl chains (as shown by the ⟨|SCH|⟩ profile in Figure 2A) while promoting a fluid-like disorder of the head group region (Figure 2B). For the POPC/diploptene bilayer, the accumulation of diploptene molecules at the mid plane of the bilayer led to substantial distortions of the leaflets, and thus, its effects on the density profiles were not analyzed in detail. The ordering of lipids was further investigated by examining how the relative alignment of pairs of POPC molecules varied with distance. This was estimated using the orientational order parameter Θ33

with temperature and comparable in magnitude in all three cases (about −7% per 20 K). As shown previously,27 BHT adopts a cholesterol-like upright orientation (Figure 2C), but its ordering effect is lower than that of cholesterol. In contrast, the behavior of diplopterol was similar to that observed previously in the case of diploptene.27 Namely, diplopterol tended to migrate in between the two leaflets as the temperature increased (Figure 2D). At 298 K, only a few diplopterol molecules moved to the core of the bilayer within the time scale of the simulations (500 ns), whereas at 343 K, almost all of the diplopterol molecules migrated to the midplane of the bilayer within 450 ns. At 363 K, the bilayer disintegrated within 200 ns, forming a mixture with water. The degree of order and distortion within the bilayer was also examined using the density profile of the phosphorus atoms along the bilayer normal (z-axis). The phosphorus density profile of each system was calculated along the z-axis over the last 200 ns of the simulation (see Supporting Information, Figure S1). The mobility along the z-axis or occupancy uP was defined as the average width of the peak for which the distribution of the phosphorus atoms along the bilayer normal is maximal. A small value of uP indicates a sharp peak, corresponding to a high degree of ordering and a relatively low range of motions of the phosphorus atoms along z. Larger uP values correspond to broad peaks and are associated with less ordering and a greater range of motions of the phosphorus atoms. This can be due to local distortion or longer-range undulations of the bilayer. Panel B in Figure 2 shows the variation of uP as a function of temperature for all of the bilayers. The value of uP for the POPC bilayer changed moderately with temperature (2.5 nm at 298 K and 2.9 nm at 343−363 K), which suggests that an increase in temperature did not enhance rippling or distortion of the bilayer. This compares favorably with atomic force microscopy measurements that showed that heating a supported DMPC (1,2dimyristoyl-sn-glycero-3-phosphocholine) bilayer up to 80 °C did not cause any topographic change beyond the gel-to-liquidcrystalline phase transition temperature.32 In the presence of

Θ=

1 ⟨3 cos2 γij − 1⟩ 2

where γij is the angle between the two vectors joining carbons 1 and 16 in the sn-1 palmitoyl chain of the POPC molecules i and j. The distance between the two lipids i and j was defined as the distance between the two phosphorus atoms. When two POPC molecules are perfectly aligned with each other (γij = 0), Θ = 1. In the case of an isotropic distribution of γij, Θ = 0.33 Figure 3 shows the Θ profile at all temperatures for all of the bilayers. As can be seen, in all cases, increasing temperature was associated with a reduction in the orientational ordering of the POPC molecules. In both the POPC/cholesterol and POPC/BHT bilayers, the degree of orientational order was more pronounced than that in the POPC bilayer. The highest degree 3955

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Figure 4. Two-dimensional RDF of the phosphorus atoms in POPC in the simulations of (A) a POPC bilayer, (B) a POPC/cholesterol bilayer, (C) a POPC/BHT bilayer, and (D) a POPC/diplopterol bilayer at 298, 323, 343, and 363 K.

suggested that their effect on ordering is less than that of cholesterol.26,27 Here, it was found that cholesterol induced a high degree of order in both the acyl chains and the head group region and that this was only weakly dependent on temperature up to 363 K. In contrast, the effect of BHT on ordering depended strongly on temperature. While BHT enhanced order of the lipid tails, its effect on the head group region was effectively negligible at high temperatures (343 and 363 K). This would suggest that BHT stabilizes the core of membranes against thermal disorder while allowing the membrane surface to remain in a fluid-like state. Given the high concentrations of BHT and BHT derivatives found in the membranes of thermophilic bacteria, this may enable the plasma membrane to remain in a lamellar phase while retaining the required degree of fluidity for the function of the membrane and membrane proteins. Diplopterol in contrast accumulated in the midplane of the bilayer in a similar manner to that observed previously for diploptene.27 This tended to decrease order in a lipid bilayer, suggesting that diploptene and diplopterol may protect membranes against environmental insults other than temperature. The simulations demonstrate that hopanoids and sterols have distinct thermotropic properties. Hopanoids stabilize the lamellar phase without adversely affecting the fluidity of membranes in thermotolerant and thermophilic bacteria.

of order was observed in the POPC/cholesterol bilayer at all temperatures. Note, in the presence of cholesterol, the variations in Θ with the distance between POPC molecules was relatively unaffected by temperature (Θ ≈ 0.7−0.9). In contrast, temperature had a significant effect on Θ in the POPC/BHT bilayer. At 298 K, orientational order within the POPC/BHT bilayer was clearly evident. However, at higher temperatures, overall ordering was significantly reduced with Θ around 0.5−0.6 at 343 and 363 K. In addition, the distinctive long-range order observed in the Θ profile at 298 K disappeared at higher temperatures. Again, this suggests that the degree of ordering within the bilayer induced by BHT is relatively constant at temperatures preferred by thermotolerant and thermophilic bacteria. As expected, the POPC/diploptene bilayer showed little, if any, order with the partitioning of diploptene between the two leaflets. Finally, positional ordering of the lipids was examined by calculating the two-dimensional radial distribution function (RDF) of the phosphorus atoms in each leaflet of all of the bilayers (Figure 4). It is clear that over the range investigated, temperature had little to no effect on the RDF of the pure POPC bilayer and only a limited effect on the POPC/ cholesterol, POPC/BHT, and POPC/diploptene bilayers. Most notably, the RDFs for the POPC/cholesterol and POPC/BHT bilayers at 298 K show the strongest short-range positional order, especially for the POPC/BHT bilayer. Interestingly, this ordering is greater than that in the POPC/cholesterol bilayer at the same temperature. The RDFs of the POPC/diploptene bilayer are similar to those of a pure POPC bilayer, indicating that the presence of diploptene in or between the leaflets has nearly no influence on the distribution of the phosphorus atoms in the plane of the leaflets. In summary, the properties of POPC bilayers containing cholesterol and the two hopanoids BHT and diplopterol were investigated at a range of temperatures that correspond to the growing conditions of thermotolerant and thermophilic bacteria such as Alicyclobacillus acidocaldarius. Hopanoids have long been assumed to promote lipid ordering in a similar fashion to cholesterol in eukaryotic membranes, but previous studies have

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ASSOCIATED CONTENT

S Supporting Information *

Details of the simulation method and force field parameters for bacteriohopanetetrol and diplopterol. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +61 (0)7 3365 7562. Fax: +61 (0)7 3365 3872. Notes

The authors declare no competing financial interest. 3956

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ACKNOWLEDGMENTS This work was funded from the National Health and Medical Research Council (Project Grant APP1044327) with the assistance of high-performance computing resources provided by the National Computational Infrastructure National Facility and iVEC located at iVEC@Murdoch through the National Computational Merit Allocation Scheme supported by the Australian Government (Projects m39 and m72).

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