Localization of trehalose in partially hydrated DOPC bilayers: insights into cryoprotective mechanisms

Abstract

Trehalose, a natural disaccharide with bioprotective properties, is widely recognized for its ability to preserve biological membranes during freezing and dehydration events. Despite debate over the molecular mechanisms by which this is achieved, and that different mechanisms imply quite different distributions of trehalose molecules with respect to the bilayer, there are no direct experimental data describing the location of trehalose within lipid bilayer membrane systems during dehydration. Here, we use neutron membrane diffraction to conclusively show that the trehalose distribution in a dioleoylphosphatidylcholine (DOPC) system follows a Gaussian profile centred in the water layer between bilayers. The absence of any preference for localizing near the lipid headgroups of the bilayers indicates that the bioprotective effects of trehalose at physiologically relevant concentrations are the result of non-specific mechanisms that do not rely on direct interactions with the lipid headgroups.

Keywords: anhydrobiology; cryobiology; cryoprotection; lipid membrane; membrane diffraction.

Figure 1.

Cartoon illustrating two potential scenarios for the location of sugars in a lipid membrane system. In (a), most of the solute is excluded from the bilayer surface; in (b), specific interactions between the sugar and lipid headgroups result in higher concentrations of sugar bound to the bilayer surface. (Online version in colour.)

Figure 2.

Rocking curve of the first-order Bragg reflection trehalose/DOPC 0.5:1 equilibrated at 57% RH over a saturated NaBr solution with 8% D₂O. The rocking curve (circles) is described by the sum (solid line) of two Gaussians (dashed and dotted lines): a sharp peak with contributions from the mosaicity of the bilayers, and a low broad peak owing to the non-lamellar lipid formations at the edges of the bilayers. The FWHM of the sharp Gaussian is 0.27° (ω). (Online version in colour.)

Figure 3.

SLD profiles (acyl group centred) showing the change in the scattering density due to the deuteration of the trehalose. As the trehalose deuteration increases (bottom to top), the only significant change is in the water layer. Vertical lines at ±17.5 Å indicate the maximum in the non-deuterated trehalose SLD profile owing to the phosphate group of the lipid headgroup. (Online version in colour.)

Figure 4.

Scattering density difference profiles (water layer centred) showing the water distribution (a) and the trehalose distribution (b). Panel (a) is the difference between measurements with 50% D₂O and 8% D₂O. Panel (b) is the difference between deuterated trehalose and hydrogenated trehalose. A single Gaussian peak fits the trehalose data, where two Gaussians are needed to fit the water distribution. Horizontal lines indicate the FWHM of the envelope of the summed Gaussians' fit to the water distribution (FWHM = 13.8 Å, a) and the FWHM of the Gaussian fit to the trehalose distribution (FWHM = 10.2 Å, b). Vertical lines at ±8.0 Å are equivalent to those in figure 3. (Online version in colour.)