Ben Kent

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

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.

Phospholipid Membrane Protection by Sugar Molecules during Dehydration-Insights into Molecular Mechanisms Using Scattering Techniques

Scattering techniques have played a key role in our understanding of the structure and function of phospholipid membranes. These techniques have been applied widely to study how different molecules (e.g., cholesterol) can affect phospholipid membrane structure. However, there has been much less attention paid to the effects of molecules that remain in the aqueous phase. One important example is the role played by small solutes, particularly sugars, in protecting phospholipid membranes during drying or slow freezing. In this paper, we present new results and a general methodology, which illustrate how contrast variation small angle neutron scattering (SANS) and synchrotron-based X-ray scattering (small angle (SAXS) and wide angle (WAXS)) can be used to quantitatively understand the interactions between solutes and phospholipids. Specifically, we show the assignment of lipid phases with synchrotron SAXS and explain how SANS reveals the exclusion of sugars from the aqueous region in the particular example of hexagonal II phases formed by phospholipids.

The inverse hexagonal - inverse ribbon - lamellar gel phase transition sequence in low hydration DOPC:DOPE phospholipid mixtures

The inverse hexagonal to inverse ribbon phase transition in a mixed phosphatidylcholine-phosphatidylethanolamine system at low hydration is studied using small and wide angle X-ray scattering. It is found that the structural parameters of the inverse hexagonal phase are independent of temperature. By contrast the length of each ribbon of the inverse ribbon phase increases continuously with decreasing temperature over a range of 50 degrees C. At low temperatures the inverse ribbon phase is observed to have a transition to a gel lamellar phase, with no intermediate fluid lamellar phase. This phase transition is confirmed by differential scanning calorimetry.

Intrinsically Disordered Stress Protein COR15A Resides at the Membrane Surface during Dehydration

Plants from temperate climate zones are able to increase their freezing tolerance during exposure to low, above-zero temperatures in a process termed cold acclimation. During this process, several cold-regulated (COR) proteins are accumulated in the cells. One of them is COR15A, a small, intrinsically disordered protein that contributes to leaf freezing tolerance by stabilizing cellular membranes. The isolated protein folds into amphipathic α-helices in response to increased crowding conditions, such as high concentrations of glycerol. Although there is evidence for direct COR15A-membrane interactions, the orientation and depth of protein insertion were unknown. In addition, although folding due to high osmolyte concentrations had been established, the folding response of the protein under conditions of gradual dehydration had not been investigated. Here we show, using Fourier transform infrared spectroscopy, that COR15A starts to fold into α-helices already under mild dehydration conditions (97% relative humidity (RH), corresponding to freezing at -3°C) and that folding gradually increases with decreasing RH. Neutron diffraction experiments at 97 and 75% RH established that the presence of COR15A had no significant influence on the structure of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. However, using deuterated POPC we could clearly establish that COR15A interacts with the membranes and penetrates below the headgroup region into the upper part of the fatty acyl chain region. This localization is in agreement with our hypothesis that COR15A-membrane interaction is at least, in part, driven by a hydrophobic interaction between the lipids and the hydrophobic face of the amphipathic protein α-helix.

Direct Comparison of Disaccharide Interaction with Lipid Membranes at Reduced Hydrations

Understanding sugar-lipid interactions during desiccation and freezing is an important step in the elucidation of cryo- and anhydro-protection mechanisms. We determine sucrose, trehalose, and water concentration distributions in intra-bilayer volumes between opposing dioleoylphosphatidylcholine bilayers over a range of reduced hydrations and sugar concentrations. Stacked lipid bilayers at reduced hydration provide a suitable system to mimic environmental dehydration effects, as well as a suitable system for direct probing of sugar locations by neutron membrane diffraction. Sugar distributions show that sucrose and trehalose both behave as typical uncharged solutes, largely excluded from the lipid bilayers regardless of sugar identity, and with no correlation between sugar distribution and the lipid headgroup position as the hydration is changed. These results are discussed in terms of current opinions about cryo- and anhydro-protection mechanisms.