pubs.acs.org/Langmuir © 2010 American Chemical Society
Water Replacement Hypothesis in Atomic Details: Effect of Trehalose on the Structure of Single Dehydrated POPC Bilayers E. A. Golovina,† A. Golovin,‡ F. A. Hoekstra,† and R. Faller*,§ †
Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands, ‡Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow, Russia, and §Department of Chemical Engineering & Materials Science, University of California, Davis, Davis, California 95616 Received March 3, 2010. Revised Manuscript Received June 4, 2010 We present molecular dynamics (MD) simulations to study the plausibility of the water replacement hypothesis (WRH) from the viewpoint of structural chemistry. A total of 256 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) lipids were modeled for 400 ns at 11.7 or 5.4 waters/lipid. To obtain a single dehydrated bilayer relevant to the WRH, simulations were performed in the NPxyhzT ensemble with hz >8 nm, allowing interactions between lipids in the membrane plane and preventing interactions between neighboring membranes via periodic boundary conditions. This setup resulted in a stable single bilayer in (or near) the gel state. Trehalose caused a concentration-dependent increase of the area per lipid (APL) accompanied by fluidizing the bilayer core. This mechanism has been suggested by the WRH. However, dehydrated bilayers in the presence of trehalose were not structurally identical to fully hydrated bilayers. The headgroup vector was in a more parallel orientation in dehydrated bilayers with respect to the bilayer plane and maintained this orientation in the presence of trehalose in spite of APL increase. The total dipole potential changed sign in dehydrated bilayers and remained slightly positive in the presence of trehalose. The model of a dehydrated bilayer presented here allows the study of the mechanisms of membrane protection against desiccation by different compounds.
Introduction It is well established that trehalose protects membranes in desiccation tolerant organisms.1,2 This “lesson from nature” is used to protect the content of dry liposomes against leakage in the pharmaceutical industry.3,4 Trehalose can also ensure survival of human blood cells during freeze-drying,5 which bears promise for blood banking. The water replacement hypothesis (WRH) describes the mechanism of membrane protection by trehalose.1,2 This mechanism is based on replacement of water molecules by sugars in their interactions with polar groups of membrane lipids. These interactions maintain spacing between lipids and prevent the increase of the membrane main gel to fluid phase transition temperature Tm. As a consequence, dry membranes remain in a fluid state at physiological temperatures and avoid a phase transition during rehydration. The transient coexistence of fluid and gel phases in a membrane during rehydration causes leakage and is detrimental for living organisms. The WRH has considerable experimental support (see e.g. refs 2 and 3 and references therein). However, all experimental *To whom correspondence should be addressed. (1) Crowe, J. H.; Crowe, L. M.; Chapman, D. Preservation of membranes in anhydrobiotic organisms - The role of trehalose. Science 1984, 223 (4637), 701703. (2) Crowe, J. H.; Hoekstra, F. A.; Crowe, L. M. Anhydrobiosis. Annu. Rev. Physiol. 1992, 54, 579-599. (3) Crowe, J. H.; Crowe, L. M.; Oliver, A. E.; Tsvetkova, N.; Wolkers, W.; Tablin, F. The trehalose myth revisited: Introduction to a symposium on stabilization of cells in the dry state. Cryobiology 2001, 43 (2), 89-105. (4) Crowe, J. H.; Crowe, L. M.; Wolkers, W. F.; Oliver, A. E.; Ma, X.; Auh, J.-H.; Tang, M.; Zhu, S.; Norris, J.; Tablin, F. Stabilization of Dry Mammalian Cells: Lessons from Nature. Integr. Comp. Biol. 2005, 45, 810-820. (5) Wolkers, W. F.; Walker, N. J.; Tablin, F.; Crowe, J. H. Human platelets loaded with trehalose survive freeze-drying. Cryobiology 2001, 42, 79-87. (6) Lee, C. W. B.; Das Gupta, S. K.; Mattai, J.; Shipley, G. G.; Abdel-Mageed, O. J.; Makriyannis, A.; Griffin, R. G. Characterization of the L-lambda phase in trehalose-stabilized dry membranes by solid-state NMR and X-ray diffraction. Biochemistry 1989, 28, 5000-5009.
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data are indirect, and there is only limited structural data on lipids that can support mechanisms described by WRH.6,7 Thus, alternative hypotheses which explain experimental data by other mechanisms than interactions of disaccharides with lipid polar groups have been proposed.8-11 They deny the role of sugar/lipid interactions and consider sugar vitrification as the main mechanism of membrane protection by trehalose at low ( 0, the PN vector points outward (projection on the bilayer normal is positive), resulting in a positive lipid potential. The total lipid potential is the sum of the z-projections of all orientations of the PN vector. Increasing populations of PN vectors with cos θ < 0 will decrease the lipid potential due to increase of the negative z-projection. We estimate the portion of inward oriented PN vectors, producing negative z-projection of the PN vector, as the area under the cumulative curve at -1 < cos θ < 0 (Figure 9, inset as an example). Indeed, we have found a negative correlation between lipid potential and inward oriented PN vectors (Figure 9). The data were fitted by a Boltzmann distribution (blue line). Goodness of fit is calculated as adjusted R2 = 0.857 98. Trehalose, Water, and Total Potentials. The water potential decreases with dehydration to a greater extent than the lipid potential (Figure 6). Thus, the water potential does not compensate for the lipid potential, and the total potential becomes slightly positive in v11-00 models and even more so in v5-00. The water (51) Pandit, S. A.; Bostick, D.; Berkowitz, M. L. Molecular dynamics simulation of a dipalmitoylphosphatidylcholine bilayer with NaClþ. Biophys. J. 2003, 84, 3743-3750. (52) Mukhopadhyay, P.; Monticelli, L.; Tieleman, D. P. Molecular dynamics simulation of a palmitoyl-oleoyl phosphatidylserine bilayer with Naþ counterions and NaCl. Biophys. J. 2004, 86 (3), 1601-1609. (53) Seelig, J. [2H] Hydrogen and [31P] phosphorus nuclear-magnetic-resonance and neutron-diffraction studies of membranes. Biochem. Soc. Trans. 1978, 6, 40-42.
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Figure 8. Distributions of PN vector orientations (cos θ) in dehydrated models without trehalose (v11-00 and v5-00, gray lines) in comparison with the fully hydrated bilayer (h28-00, black line). The distributions were fitted with two (A, v11-00) and four (B, v5-00) Gaussian functions (in separate colors). Red: sum of all fitted curves.
Figure 9. Correlation between population of inward oriented PN vectors (cos θ < 0) and lipid potential. Data represent all the models: black stars are models without trehalose and with different water contents (indicated by arrows); red circles are models with different trehalose concentration at water content of 11.7 waters/ lipid; blue triangles are models with different trehalose concentration and 5.4 waters/lipid. The data are fitted by Boltzmann function (blue line). Goodness of fit is calculated as adjusted R2 = 0.857 98. The population of inward oriented PN vectors is calculated as area under the cumulative curve of PN distribution over cos θ. The way of calculation is shown in the inset for v5-10 as an example. In the inset the distribution of PN vector orientations with fitted Gaussian functions are shown in color. The cumulative curve is shown as a gray line. The area under the gray line at cos θ < 0 represents the total number of PN vectors oriented inward the bilayer. Because the total lipid potential is the sum of the z-projections of all orientations of the PN vector, increasing populations of PN vectors with -1 < cos θ < 0 will decrease the lipid potential due to increase of the negative z-projection.
potential becomes less negative with trehalose, and this effect is concentration dependent (Figure 7). Trehalose also causes a negative potential itself (model v11-10 as example in Figure 7 inset) on top of the negative potential Langmuir 2010, 26(13), 11118–11126
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of water. The absolute value of the trehalose potential slightly increases with increasing of trehalose:lipid ratio (Figure 7). Water and trehalose potentials together cannot compensate for even the decreased lipid potential, and the total potential remains slightly positive in all cases except v5-14 (Figure 7).
Discussion In this paper we present results of MD simulations of a single POPC bilayer at intermediate (11.7 waters/lipid) and low (5.4 waters/lipid) hydration. The separation of lipid bilayers was provided by a 4 nm layer of empty space, which we call vacuum. The necessity of separation of dehydrated lipid bilayers became evident when we failed to obtain the gel state in MD simulations of POPC bilayer stacks even at 5.4 waters/lipid,24 while experimentally a gel state of dehydrated PC bilayers is well established. We expect that the inconsistency between MD simulations and experimental data results largely from periodic boundary conditions. There are, however, a number of secondary effects like the system size (which limits fluctuations and therefore changes fluctuation dependent quantities), the force field, and several others. For a more detailed discussion we refer the reader to a recent contribution by Poger et al.54 When the water layer in the interbilayer space is absent, the interfaces of neighboring bilayers come into close contact, which might have two consequences. First, water of the hydration shell is shared by both bilayers. Second, interfaces of adjacent bilayers can interpenetrate and cause self-spacing. The first factor would result in an effective almost doubling of water content, and 5.4 waters/lipids in fact can be considered close to 10.8 waters per lipid. However, even the further decrease of water content to 2 waters/lipid did not result in formation of the gel phase. We have shown that interpenetration and self-spacing of two adjacent bilayers in dehydrated stack bilayers may be one of the main reasons of the absence of a gel state of lipid acyl chains at low water content.24 The headgroups overlap is considered as the main problem in APL calculations in stack bilayers at water contents less than 12 waters/lipid in X-ray experiments.28 To prevent overlap, the interfaces have to be separated. There are two requirements for the separating medium: it should not interact with bilayers, and the separation has to be large enough to cancel or at least significantly weaken the interactions between bilayer interfaces. We have found that in our case 4 nm (2 nm from each leaflet) of empty space (vacuum) between bilayers is enough to exclude interactions; the actual value for the vacuum layer will depend on the details of the simulations, most notably the cutoff and the implementation of the electrostatics. The ensemble NPT was converted to NhzpxyT, where the constant pressure of 1 atm was maintained only in the xy-plane, while in the z-direction the height of the box was fixed and was big enough to prevent lipid polar group interactions via PBC. In such a model the absence of interactions between bilayers along the z-axis and maintaining interactions in the xy-plane results in a stable single bilayer (400 ns of simulation) in (or near) a gel state both at 5.4 and 11.7 waters/lipid. The (near) gel state of dehydrated POPC bilayers has been concluded from low APL (0.485 nm2 for v5-00 and 0.515 nm2 for v11-00, Table 2) accompanied by an increased order parameter for all carbon atoms (Figure 2), an increased PP separation, and more pronounced methyl trough (Table 3, Figure 4). (54) Poger, D.; Mark, A. E. On the Validation of Molecular Dynamics Simulations of Saturated and cis-Monounsaturated Phosphatidylcholine Lipid Bilayers: A Comparison with Experiment. J. Chem. Theory Comput. 2010, 6 (1), 325-336.
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Our dehydrated single bilayer model is different from the one proposed by Leekumjorn and Sum,30 where dehydrated DPPC (10 waters/lipid) bilayers lost structural integrity within 30 ns of simulation. Formation of nonbilayer structures of dehydrated DPPC is not supported by experimental data. It is well established that three methyl groups on the nitrogen of PC lipids contribute a steric component to prevent this lipid from forming nonbilayer structures.32 The lamellar phase reappears even in PE lipids at sufficiently low hydration.32 The reported formation of nonbilayer structures of DPPC at 10 waters/lipid30 probably results from an unusual setup of MD simulations. These authors fixed the size of the xy area of a dehydrated bilayer to the value of hydrated bilayers (APL 0.645 nm2) under zero lateral compressibility (NPzAT ensemble). Such conditions prevent lateral interactions between lipids resulting in destabilization of the bilayer. In our model, using the NPxyhzT ensemble, we allow lipid interactions within xy-plane in a dehydrated bilayer. As a result, we obtained a bilayer in a gel state, which was stable within 400 ns. The aim of this work was to study the water replacement hypothesis (WRH) in atomic details. WRH is based on four main statements: (1) dehydrated membranes are in a gel state; (2) rehydration of the membrane in the gel state causes leakage, which is detrimental for membrane integrity; (3) trehalose interacts with dehydrated lipids and prevents gel phase formation; and (4) rehydration of membranes in fluid state does not cause leakage. Using empty space between lipid lamellae, we obtained dehydrated single bilayers in a gel state, which fits the first statement of the WRH. This model can be further used to study the validity of the third statement of the WRH, claiming that trehalose increases the spacing between lipids and promote fluid state of a dehydrated bilayer. Here, we call a bilayer dehydrated if there is no interbilayer water present. This occurs when the water content is below the size of the hydration shell (around 12 waters/lipid). We have shown that trehalose increases the APL of dehydrated POPC bilayers (at both 11.7 and 5.4 waters/lipid) in a concentration-dependent manner (Figure 1, Table 2). Increasing APL correlates with decreasing order parameter (Figure 3), PP separation and depth of the methyl trough (Table 3). These structural data are consistent with gradual concentration-dependent fluidization of bilayer core resulted from interactions between lipids and trehalose. Therefore, our model confirms the third statement of the WRH as well. However, a dehydrated bilayer in the presence of trehalose is structurally different from a fully hydrated one in spite of a similar degree of fluidization of the membrane core (Figure 5, Table 3). Even at trehalose concentration of 1.4 trehalose/lipid the APL does not reach the value of the fully hydrated bilayer in both v11 and v5 models. At this concentration trehalose has a “saturation” effect on membrane phase transition according to calorimetric data.55 The order parameter also remains higher than in the hydrated bilayer (Figures 2 and 3). Contrary to this, some experiments show overfluidization of dry DPPC bilayers in the presence of trehalose (DPPC:trehalose=1:2).6,7 According to 2H NMR data, acyl chains are much more disordered in dehydrated DPPC: trehalose mixtures (1:2) above the phase transition than in a fluid state of hydrated DPPC bilayer. The authors call this new type of fluid state a λ-phase.7 On the other hand, Quinn et al.29 did not find any structural evidence of a new fluid state designated as λ-phase studying dehydrated trehalose/DPPC (2:1) mixture by a real time X-ray diffraction. In our model acyl chains remain more (55) Tsvetkov, T. D.; Tsonev, L. I.; Tsvetkova, N. M.; Koynova, R. D.; Tenchov, B. G. Effect of trehalose on the phase properties of hydrated and lyophilized dipalmitoylphosphatidylcholine multilayers. Cryobiology 1989, 26, 162-169.
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ordered in dehydrated POPC (both at 11.7 and 5.4 waters/lipid) at 1.4 trehalose/lipid at 310 K in comparison with the fully hydrated bilayer, which is in a fluid phase at the same temperature (Figure 3). This is consistent with the lower APL in comparison with the fully hydrated bilayer (Table 2). Therefore, our model showed the trend of increasing APL of dehydrated bilayer by trehalose, but we failed to obtain the structural characteristics similar to that of the hydrated bilayer even in the excess of trehalose, when sugar forms a phase outside of the lipid headgroup area (Figure 5). More structural differences between fully hydrated POPC and dehydrated POPC/trehalose mixtures are observed in the headgroup region. The average orientation of the PN vector is shifted parallel to the membrane interface with dehydration and remains like that with trehalose (Figures 8 and 9) consistent with NMR and neutron diffraction experiments. NMR spectra of selectively deuterated headgroups of PC lipids have shown that dehydration results in the choline group aligning more closely with the bilayer surface.56,57 Neutron diffraction indicated a spatial limit for PN vector reorientation during bilayer dehydration of 12°.56 67% w/v of trehalose at 293 K was shown to cause the shift of PN to a more parallel orientation than in the hydrated POPC bilayer without trehalose.58 A similar effect of trehalose on PN orientation might be expected in a dehydrated bilayer. The shift of the average PN orientation in dehydrated bilayer results in the increased proportion of inward oriented PN vectors (Figure 9), which decreases the z-component of the lipid dipole and, consequently, the lipid potential (Figure 6). The absolute value of water potential decreases with dehydration (Figure 6) and decreases further with trehalose (Figure 7) probably due to the migration of some water molecules from the phosphate group to trehalose (Figure 5). Although trehalose creates a negative potential, both negative potentials of trehalose and water cannot compensate for the decreased lipid potential, and the total potential remains slightly positive in dehydrated bilayer at all trehalose concentrations, while the total potential in hydrated POPC bilayers (h28-00) is negative, -0.46 V.24 The value of the total potential of hydrated POPC bilayer is in agreement with the experimental data on dipole potential (400 mV)59 but higher than that in bilayers (220-280 mV).60 The values of the dipole potential obtained in MD are usually higher than experimental values (around 600 mV).61 This is likely rooted in the simulation assumption that the dielectric constant is fixed at ε = 1. Simulations of dry DOPC bilayers have shown the decrease of the total potential from 500 mV at 16 waters/lipid to -300 mV at 11.4 waters/lipid and þ400 mV at 5.4 waters/lipid.27 This is in agreement with our observation of changing the total potential with dehydration. In the model here the total potential was -460, þ120, and þ200 mV at 28.5, 11.7, and 5.4 waters/lipid, respectively. Because experimentally the dipole potential can be obtained for fully hydrated bilayer only, we cannot compare our (56) Bechinger, B.; Seelig, J. Conformational changes of the phosphatidylcholine headgroup due to membrane dehydration. A 2H-NMR study. Chem. Phys. Lipids 1991, 58, 1-5. (57) Ulrich, A. S.; Watts, A. Molecular response of the lipid headgroup to bilayer hydration monitored by 2H-NMR. Biophys. J. 1994, 66, 1441-1449. (58) Bechinger, B.; Macdonald, P. M.; Seelig, J. Deuterium NMR studies of the interactions of polyhydroxyl compounds and of glycolipids with lipid model membranes. Biochim. Biophys. Acta 1988, 943, 381-385. (59) Brockman, H. Dipole potential of lipid membranes. Chem. Phys. Lipids 1994, 73, 57-79. (60) Clarke, R. J. The dipole potential of phospholipid membranes and methods for its detection. Adv. Colloid Interface Sci. 2001, 89-90, 263-281. (61) Berkowitz, M. L.; Bostick, D. L.; Pandit, S. Aqueous solutions next to phospholipid membrane surfaces: insights from simulations. Chem. Rev. 2006, 106, 1527-1539.
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results on dehydrated bilayers with experimental data. However, some support comes from data by Luzardo et al.62 where a concentration-dependent decrease of the lipid potential of DMPC monolayers in the presence of trehalose has been observed. The inversion of the sign of the total dipole potential in dry bilayers with and without trehalose might provide a new mechanism of membrane protection against dehydration by some proteins. Late embryogenesis abundant (LEA) proteins have been proposed to contribute toward desiccation tolerance, but the actual mechanism of action is unclear.63 Yeast HSP 12 was first identified as a putative heat shock protein but later has been classified as a LEA-like protein.64 Membrane protection by HSP 12 against dehydration was only observed with positively charged liposomes and not with either neutral or negatively charged liposomes.65 Therefore, inversion of the sign of total dipole potential of a bilayer during dehydration from negative to slightly positive might provide the conditions for interactions with the LEA-like proteins and thus stabilize dehydrated membranes. The data presented in this study show that the separation of dehydrated bilayers by empty space in MD simulations provides a means to study of the mechanisms of membrane protection against desiccation by different compounds. This model is experimentally relevant for two reasons. First, POPC bilayer is stable in a gel state at a low water content at ambient temperature, which is in agreement with all experimental data on PC dry bilayers. Second, the area per lipid of dry POPC increases when trehalose is added. Although there is no direct experimental data on APL values of a dry bilayer with trehalose, the decrease of the phase transition temperature Tm of dry PC bilayer in the presence of trehalose is commonly used as an indicator of the increased APL.66 Area per lipid, related order parameter, and density profiles are the only structural data which are influenced by the model. Other structural data (PN vector orientation and potential) were similar in both stack and vacuum models, so the comparison with the experiments, which were carried out on multilamellar liposomes, is still valid for such data. Our vacuum model partly validates the WRH by showing that dehydration causes the decrease of APL and gel-state formation, and trehalose increases APL and fluidizes the core of the dry bilayer. However, the detailed structure of dry POPC bilayer in the excess of trehalose is different from a fully hydrated bilayer, particularly in the headgroup region. Acknowledgment. This work was partly supported by project no. 10195 from the Dutch Foundation for Technological Research STW (E.A.G.) and partly by the NATO Science Program (NATO collaborative linkage grant LST.CLG.980168 (A.V.G. and F.A.H.). Computer resources were provided by the Research Computing Center of Moscow State University. The supercomputer “Chebyshev” was used for all modeling studies. (62) Luzardo, M. C.; Amalfa, F.; Nunez, A. M.; Diaz, S.; Lopez, A. C. B.; Disalvo, E. A. Effect of trehalose and sucrose on the hydration and dipole potential of lipid bilayers. Biophys. J. 2000, 78, 2452-2458. (63) Chakrabortee, S.; Boschetti, C.; Walton, L. J.; Sarkar, S.; Rubinsztein, D. C.; Tunnacliffe, A. Hydrophilic protein associated with desiccation tolerance exhibits broad protein stabilization function. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (46), 18073-18078. (64) Mtwisha, L.; Brandt, W.; McCready, S.; Lindsey, G. G. HSP 12 is a LEAlike protein in Saccharomyces cerevisiae. Plant Mol. Biol. 1998, 37, 513-521. (65) Sales, K.; Brandt, W.; Rumbak, E.; Lindsey, G. The LEA-like protein HSP 12 in Saccharomyces cerevisiae has a plasma membrane location and protects membranes against desiccation and ethanol-induced stress. Biochim. Biophys. Acta 2000, 1463, 267-278. (66) Hoekstra, F. A.; Wolkers, W. F.; Buitink, J.; Golovina, E. A.; Crowe, J. H.; Crowe, L. M. Membrane stabilization in the dry state. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 1997, 117 (3), 335-341.
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