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* Biochemistry Department, Ultrafast Laser and Spectroscopy Laboratory, and Molecular Dynamics Group, Groningen Biomolecular Science and Biotechnology Institute and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
Correspondence: Address reprint requests to Bert Poolman, Tel.: 31-50-363-4190; Fax: 31-50-363-4165; E-mail: b.poolman{at}rug.nl.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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) accumulate disaccharides in response to various stresses, such as temperature (2), osmotic (3), and oxidative stress (4). Upon cold and drought stress, many organisms accumulate trehalose to protect both proteins and lipid membranes (see Oliver et al. (5) and Crowe et al. (6) for reviews). Higher plants often accumulate sucrose instead of trehalose (7). The protection of biological structures by sugars has applications in a wide range of fields, including food preservation and cryoconservation of eukaryotic cell lines (8). Recently, we showed that sucrose and trehalose (Fig. 1) protect membrane proteins from inactivation upon the conversion of large unilamellar vesicles (LUVs) to giant unilamellar vesicles (GUVs), which involves a cycle of dehydration and rehydration (9).
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,6). Drying of membranes, composed of lipids with a low phase transition temperature (Tm), induces a transition from the liquid crystalline to the gel phase. This causes solute leakage, membrane fusion, and aggregation of membrane proteins. The protective effects of sugars are twofold: i), the formation of a glassy matrix, and ii), direct interactions between the lipids and the sugars (10–12). With a glassy matrix, the sugars form a hydration layer of amorphous glasses, preventing mechanical disruption and denaturation of (membrane) proteins. The formation of a glassy matrix is related to the glass transition temperature (Tg) of the sugars, and sugars with a high Tg generally provide better protection than sugars with a low Tg (7,10,11). Dehydration of membranes increases the Tm of the lipids and causes the liquid crystalline to gel phase transition, which has consequences for the membrane as permeability barrier and the conformational state of the embedded proteins. In the dry state, hydrogen-bond formation between the sugar molecules and the lipid headgroups reduces this increase of the Tm and thereby prevents the phase transition (6,10–12).
Of all tested sugars, trehalose (Tg = 106°C (13)) has been shown to cause the largest suppression of the Tm, offering the largest protection of vesicles and cells against solute leakage and fusion (5,6). However, disaccharides like sucrose (Tg = 60°C (13)) have been shown to also offer good protection against these membrane-rupturing events (14,15). In general, fructans provide better protection against solute leakage than glucans, whereas glucans provide better protection against membrane fusion (16). Both the suppression of the Tm and the formation of a glassy matrix are necessary for preventing membrane fusion, whereas the glassy matrix appears sufficient for the prevention of contents leakage from vesicles (11,16). Recently, it was found that the sugar/lipid ratios needed to prevent membrane fusion were 10-fold higher than those needed to suppress the Tm, and even higher ratios were needed to prevent solute leakage (12). Thus, despite a large number of studies on the protective effects of sugars on membranes, the precise interactions of these sugars with the lipids are poorly understood.
Most studies on the interactions between sugars and phospholipid membranes focus either on the Tm or on the protection against solute leakage and fusion of liposomes or whole cells, and little is known about the interactions between sugars and the lipids in the fully hydrated state. In solution, trehalose interacts directly with phospholipid bilayers, as was shown with Fourier transform infrared spectroscopy (17). These interactions must differ from the interactions in the dry state, because trehalose increased the Tm of fully hydrated lipid membranes in solution but not of membranes in the dried state (18,19). Here, we report on sugar-membrane interactions, using fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. FCS was used to measure the influence of different sugars on the lateral diffusion of fully hydrated lipids in GUVs. The MD simulations were used to rationalize the observed differences in diffusion coefficients.
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METHODS |
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,2-dioleoyl-sn-glycero-3-phosphatidylcholine) or a mixture of DOPC and DOPS (1,2-dioleyl-sn-glycero-3-phosphatidylserine, Avanti Polar-Lipids, Alabaster, AL) at a 3:1 molar ratio. For FCS, the fluorescent lipid analog 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO; Invitrogen, Carlsbad, CA; excitation and emission wavelengths of 484 and 499 nm, respectively) was incorporated at a DiO/lipid ratio of 1:10,000 molar.
Fluorescence correlation spectroscopy
FCS measurements were carried out on a laser scanning confocal microscope (9), based on an inverted microscope Axiovert S 100 TV (Zeiss, Jena, Germany) in combination with a galvanometer optical scanner (model 6860, Cambridge Technology, Watertown, MA) and a microscope objective nanofocusing device (P-721, Physik Instrumente, Karlsruhe/Palmbach, Germany). For excitation of the fluorescent lipid analog DiO, an argon ion laser (488 nm, Spectra-Physics, Mountain View, CA) was focused by a Zeiss C-Apochromat infinity-corrected 1.2 numerical aperture 63x water immersion objective. The intensity of the laser beam did not exceed 10 µW at the back aperture of the objective. Emission was collected through the same objective, separated from the excitation beam by a beam pick-off plate (BSP20-A1, ThorLabs, Newton, NJ) and directed through an emission filter (HQ 535/50, Chroma Technology, Rockingham, VT) and a pinhole (diameter of 30 µm) onto an avalanche photodiode (SPCM-AQR-14, EG&G, Albuquerque, NM). The fluorescence signal was digitized, and the autocorrelation curve was calculated using a multiple 
algorithm. The setup was calibrated by measuring the known diffusion coefficient of Alexa fluor 488 in water (Invitrogen; D = 300 µm2 s–1). The lateral radius 
xy, defined as the point where the fluorescence count rate per molecule decreased e2 times, was 180 nm, corresponding to a detection volume of 
0.20 fl.
For the FCS measurements, the focal volume was positioned at the upper pole of a GUV, as described in Doeven et al. (9). For each sample, the fluorescence autocorrelation of 10 GUVs was measured for 80 s. The fluorescence autocorrelation curves were fitted with a model for two-dimensional Brownian motion (20). The viscosities of the sugar solutions were determined by measuring the diffusion constant of free Alexa fluor 488 in the various solutions. The diffusion constant is linearly related to the viscosity, as described by the Einstein-Stokes model.
General atomistic MD simulation conditions
All MD simulations at atomistic scale were performed using the GROMACS code (21), with parameters based on the GROMOS force field parameter set 53A6 (22). The simple point charge water model (23) was used to model water. Newton's equations of motion were integrated using the leapfrog algorithm (24) with a 2-fs time step. The LINCS method (25) was applied to constrain all bond lengths. The water geometry was constrained using the SETTLE algorithm (26). The simulations were carried out in a rectangular box with an isothermal-isobaric (NPT) ensemble at 300 K, using a Berendsen thermostat (27), with a coupling constant of 0.1 ps. The pressure was coupled semiisotropically using a Berendsen scheme (27) with a reference pressure of 1 bar in all directions, a relaxation time of 1 ps, and an isothermal compressibility of 5 x 10–5 bar–1.
The nonbonded interactions were calculated using a twin-range cutoff scheme. All Lennard-Jones and electrostatic interactions within the 0.9-nm short-range cutoff were evaluated every time-step, based on a pair list recalculated every 10 steps. The Lennard-Jones potentials and electrostatic interactions within the long-range cutoff distance of 1.4 nm were calculated simultaneously with each pair list update and assumed constant in between. Electrostatic interactions beyond the 1.4-nm cutoff radius were corrected with a reaction field potential, with 
r = 62 (28). For analysis, the atomic coordinates were saved every 50 ps. The MD data were analyzed using the standard GROMACS tools (21) as described in De Vries et al. (29) and Pereira et al. (30).
Used topologies
The topologies of both sucrose (ß-D-fructofuranosyl-
-D-glucopyranoside) and trehalose (-D-glucopyranosyl--D-glucropyranoside) for the atomistic MD simulations were generated from protein data bank files (31), using the Dundee PRODRG2 server (32). The force field parameters to describe trehalose were modified according to Lins and Hünenberger (33). The force field parameters to describe sucrose were derived starting from the force field parameters of hexopyranose-based carbohydrates (33) with respect to bond angle bending, bond stretching, dihedral deformation, improper dihedral deformation, and van der Waals interactions. For the partial charges, the classical electrostatic potential outside the sucrose molecule was fitted to the corresponding quantum-mechanical potential. Redistribution of charges was required to permit the definition of neutral charge groups with restricted sizes within the molecule, similar to that in Lins and Hünenberger (33). During the 10-ns simulations, both sugars stayed in solution up to 1.5 M, as expected from the solubility of the sugars. The topology files are provided in the Supplementary Material.
Description of initial conditions and systems
The starting conditions for the atomistic MD simulations of trehalose and sucrose with DOPC bilayer were created by deleting the water molecules from a system with an equilibrated DOPC bilayer (29). The DOPC bilayer consisted of two monolayers of 32 lipids each. A total of 16, 32, or 60 trehalose or sucrose molecules were added to this system, and the box was filled with water molecules, resulting in sugar concentrations of 0.4, 0.8, and 1.5 M, respectively. The final lipid/water ratio was higher than 1:25 molal, so the lipid bilayer was fully hydrated. The systems were equilibrated for 10 ns at 300 K, after which most of the sugars were adsorbed on the bilayer interface with the lipid headgroups, and these systems were taken as starting conditions for further simulations.
Coarse-grained MD simulations
All coarse-grained MD simulations were performed using the GROMACS code (21). Newton's equations of motion were integrated using the leapfrog algorithm (24) with a 5-fs time step. The bonds and angles were represented by a harmonic potential. The simulations were carried out in a rectangular box of fixed size (25 x 25 x 10 nm) with the temperature at 325 K, using a Berendsen thermostat (27), with a coupling constant of 0.1 ps. The Lennard-Jones potentials and forces were calculated using the shift potential implemented in GROMACS (21), which decreases potentials and forces smoothly to zero at the cutoff of 1.2 nm (34). A twin-range cutoff scheme was used with a short-range cutoff of 0.9 nm, and a long-range cutoff of 1.2 nm, with a neighbor list that was updated every 10 time steps. For analysis, the atomic coordinates were saved every 50 ps.
The lipids were represented by rods consisting of two beads and the sugars as squares consisting of four beads, with masses of 72 amu per bead. The length of all bonds between the beads was 0.47 nm with a 1250 kJ mol–1 nm–2 harmonic force constant. The interactions between molecules were either attractive or semiattractive, where the Lennard-Jones parameters were 
and 
for the attractive interactions and 
and 
for the semiattractive interactions. The interactions between lipid molecules and between sugar molecules were set to semiattractive. The interaction between each of the beads of the sugar molecules with the lipid molecules was varied from attractive to semiattractive. All interaction sites were uncharged. The lipids and the sugars were kept in two planes using positional restraints in the direction perpendicular to the planes with force constants 1000 kJ mol–1 nm–2 and 100 kJ mol–1 nm–2, respectively. The planes of the lipids and of the sugars were 0.5 nm apart. A total of 1024 lipid molecules and 512 sugar molecules was simulated for 500 ns.
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RESULTS |
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,35) and pulsed field gradient NMR (36). Addition of sugars up to 2 M reduced the lateral mobility in a concentration-dependent manner; the maximal decrease in D was 2.5-fold and observed for sucrose. For all concentrations, sucrose inhibited the diffusion of lipids more than maltose and trehalose (Fig. 2 a). This larger inhibition of the phospholipid diffusion by sucrose was observed both in membranes consisting of a 3:1 molar ratio of DOPC/DOPS and in membranes consisting of pure DOPC (not shown). The decrease by sugars could not be fully attributed to an increase in the viscosity of the bulk phase as predicted by Saffman and Delbrück (Fig. 2 b) (37).
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MD simulations
MD simulations at an atomistic scale were performed in an attempt to rationalize the differences in the interactions between sucrose and trehalose with the lipid membrane. Seven simulations were performed: a DOPC bilayer in water without sugar and DOPC bilayers in water with 0.4, 0.8, and 1.5 M sucrose or trehalose, all at 300 K. After 10-ns equilibration time, all simulations were performed for 100 ns. During the 10-ns equilibration time, a steady state was reached (not shown), in accordance with published data (30,38,39). The projected area per lipid during the simulations was independent of the sugar concentrations and was 58.4 ± 0.8 
2. This value is lower than experimental values for pure DOPC of 59.4 (40) to 72.2 ± 0.5 2 (41,42). The area per lipid is known to be lower in comparison to experimental ones when using the GROMOS 53A6 force field (22,43). Since the lipids were still in the liquid crystalline phase, the simulations were used to provide qualitative information regarding the interactions between the lipid headgroups and the sugars.
Fig. 3 shows a snapshot of the 0.8-M sucrose system after 100 ns of simulation time. It shows that most of the sucrose molecules interact with the lipid headgroups, leading to an increase in the surface concentration. The density profiles perpendicular to the membrane of different atom groups for the 0.8-M sucrose and trehalose systems are presented in Fig. 4. The density profiles show that both sucrose and trehalose were adsorbed at the DOPC bilayer interface. No differences in protrusion were observed between sucrose and trehalose or between the glucose and fructose moieties of sucrose.
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), with the sugars interacting in a similar way as in the simulations where a DOPC bilayer was present from the start. This indicates that the interaction is not an artifact of the starting conditions or the timescale of the simulation.
In the absence of sugar, a lateral diffusion constant for the lipids of D = 4.7 ± 3.2 µm2s–1 was obtained, which is in good agreement with the measured value of D = 6.5 ± 0.6 µm2s–1. The distribution of the diffusion constants of the individual lipids was wide, as can be seen in the histogram in Fig. 5, and is also reported in De Vries et al. (29). If the simulation time is increased, the diffusion constants should converge to the same value. However, MD simulations of DPPC bilayers showed only a small narrowing of the spread of the diffusion constants between 100- and 500-ns simulation (data not shown). Because of this broad distribution, it was not possible to calculate D reliably from the trajectories, and the data had to be interpreted qualitatively. It is clear that both sucrose and trehalose reduced the lateral mobility of the lipids at all three concentrations (Fig. 5). Due to the qualitative nature of the results, however, no statistically significant difference between sucrose and trehalose could be observed in the simulations.
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10% more hydrogen bonds per sugar with lipid headgroups than trehalose (Fig. 6 a). The lifetime of the hydrogen bonds did not significantly differ between sucrose and trehalose. The average number of mutual hydrogen bonds between the sugar molecules was not different for sucrose and trehalose (Fig. 6 a). Most hydrogen bonds between sugars and lipid headgroups were formed with the phosphate oxygens. The average number of sugar molecules that formed hydrogen bonds with a lipid was significantly larger for sucrose than for trehalose (Fig. 6 b), and the average number of lipids that formed hydrogen bonds with a sugar was also larger (Fig. 6 c). For the 0.8-M sugar concentrations, the distribution of the number of lipid molecules bound per sugar is shown in Fig. 6 d. In summary, sucrose interacted with more lipid molecules at the same time as trehalose.
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DISCUSSION |
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), which describes the relationship between lateral diffusion and the viscosity of the bulk phase, fails. Furthermore, one of the main assumptions of the model is that the membrane forms a homogeneous two-dimensional medium and that the radius of the molecules the membrane consists of are infinitely smaller than the radius of the diffusing particles. In the case of lipids, this assumption is obviously not satisfied, and the Saffman-Delbrück model has inherent limitations (45).
The decrease of the lateral phospholipid mobility by sucrose was also larger than that of the trisaccharide maltotriose and the tetrasaccharide maltotetrose, which have more hydrogen-bonding capabilities. Maltotriose is known to protect membranes against leakage upon freezing and drying (19,46). The stronger decrease in the lateral mobility by sucrose than that by other sugars is unlikely to be an artifact due to the use of the fluorescent lipid analog DiO, since the analog NBD C6-HPC showed a similar effect (9). NBD C6-HPC has a fluorescent moiety in the lipid tail rather than the headgroup like DiO. Furthermore, the measured effect was present in membranes consisting of pure DOPC and of a mixture of DOPC and DOPS.
The large decrease of the lateral mobility by sucrose has not been reported previously. Two experimental studies regarding the effect of sugars on the diffusion of lipids have been published (47,48), both using the alcohol sugar glycerol. A fluorescence recovery after photobleaching (FRAP) study showed that the diffusion constant decreased linearly with the sugar concentration (47), whereas an excimer formation study showed a much smaller effect, 2-fold (48). For glycerol, we measured an inhibition of the lateral phospholipid mobility of 1.5-fold (not shown), which is in agreement with the excimer formation study (48). In the FRAP study (47), a fluorescent-labeled transmembrane lipid was used in multilayer lipid sheets, and this configuration might have influenced the diffusion of the lipid probe. Neither of these studies explored sucrose or trehalose.
MD simulations
MD simulations by Pereira et al. (30) and recently Skibinsky et al. (38) showed interactions between trehalose and lipid headgroups similar to our observations, but sucrose was not investigated. An MD study, where both sucrose and trehalose were included, was performed by Sum et al. (39). They showed that both trehalose and sucrose inserted into the bilayer and interacted with multiple lipid molecules simultaneously, which is in agreement with our simulation. However, due to the length of these simulations (10 ns), no significant diffusion constant of the phospholipids could be calculated. Furthermore, the hydrogen bonds were not analyzed to the same extent as in our work. Therefore, at the start of this project, no model was available to explain the effect of different sugars on the lateral lipid mobility. We ran longer (100-ns) simulations to be able to model the influence of the sugars on the lipid headgroups. The lipid bilayers were fully hydrated so we could compare our FCS measurements with the MD simulations.
Our MD simulations at atomistic scale show that both sucrose and trehalose slow down the lateral diffusion of the lipids, although no difference between the sucrose and the trehalose was observed due to the limited timescale of the simulations. A more extensive analysis of the MD data showed that sucrose formed 10% more hydrogen bonds with phospholipid headgroups than trehalose (Fig. 6), whereas the lifetimes of the hydrogen bonds were similar. Furthermore, sucrose interacted with more lipid headgroups simultaneously than trehalose. Coarse-grained model simulations showed that an increased cross-linking of lipids by sugars can result in a relatively large reduction of the diffusion constant (Fig. 7). Based upon these observations, it is concluded that sucrose is more efficient in cross-linking the lipid headgroups than trehalose. The result is a stronger reduction of the lateral lipid mobility and provides an explanation for the FCS data.
In summary, using FCS we showed that sucrose decreases the lateral mobility of lipids more than trehalose. Atomistic and coarse-grain MD simulations provide an explanation for the differences these sugars exert on the lateral mobility of lipids. Relatively small differences in the interactions between sugars and phospholipids result in relatively large effects on the lipid mobility. These different interactions may also lead to differences in membrane protection.
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ACKNOWLEDGEMENTS |
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Submitted on August 31, 2006; accepted for publication November 9, 2006.
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REFERENCES |
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), trehalose (
), and maltose (
), and the bulk concentrations of (c) sucrose (
), and fructose (
), and (d) sucrose (
), and maltotetrose (
) are plotted.
) and fructose (
), or 1.5 M (
) trehalose.
