Unraveling the Molecular Mechanisms of Trehalose-Mediated Protection and Stabilization of Escherichia coli Lipid Membrane during Desiccation.

Trehalose, a disaccharide renowned for its ability to stabilize biomolecular architectures under strenuous conditions, finds extensive use in the cryopreservation of probiotics. A profound comprehension of its molecular-level interactions is of great significance. It is notable that current research in the realm of lipid-sugar interactions primarily employs single-component lipid bilayers, which are far from being representative of real cell membranes. Our investigation, however, utilizes molecular dynamics simulations to delve into the specifics of a realistic Escherichia coli membrane that encompasses a diverse array of lipid types, comprising fourteen distinct species, subject to varying hydration levels. The results of our study showcase that the reduction of hydration levels induces lipid ordering and the formation of gel phases, yet trehalose, by forming hydrogen bonds with lipid headgroups, serves to uphold fluidity and supplant the role of water. Moreover, our findings evince that augmented trehalose concentrations lead to a slowdown in lipid motion and contribute to the maintenance of fluidity by way of endowing a viscous matrix. It is noteworthy that our conclusions lend support to the notion that water replacement and vitrification, despite their seemingly disparate nature, need not be considered mutually exclusive in a real bacterial membrane.

How Do Cyclopropane Fatty Acids Protect the Cell Membrane of Escherichia coli in Cold Shock?

The cyclopropanation of unsaturated lipid acyl chains of some bacterial cell membranes is an important survival strategy to protect the same against drastic cooling. To elucidate the role of cyclopropane ring-containing lipids, we have simulated the lipid membrane of Escherichia coli (E. coli) and two modified membranes by replacing the cyclopropane rings with either single or double bonds at widely different temperatures. It has been observed that the cyclopropane rings provide more rigid kinks in the lipid acyl chain compared to the double bonds and therefore further reduce the packing density of the membrane and subsequently enhance the membrane fluidity at low temperatures. They also inhibit the close packing of other lipids and deleterious phase separation by strongly interacting with them. Therefore, this study has explained why E. coli bacterial strain, susceptible to freezing environments, relies on the cyclopropanation of an unsaturated chain.

Can Urea and Trimethylamine-N-oxide Prevent the Pressure-Induced Phase Transition of Lipid Membrane?

Organisms dwelling in ocean trenches are exposed to the high hydrostatic pressure of ocean water. Increasing pressure can alter the membrane packing density and fluidity and trigger the fluid-to-gel phase transition. To combat environmental stress, the organisms synthesize small polar solutes, which are known as osmolytes. Urea and trimethylamine-N-oxide (TMAO) are two such solutes found in deep-sea creatures. While TMAO stabilizes protein, urea induces protein denaturation. These solutes strongly influence the packing density and membrane fluidity of the lipid bilayer at different conditions. But can these solutes affect the pressure-induced phase transition of the lipid membrane? In the present work, we have studied the effect of these two solutes on pressure-induced fluid-to-gel phase transition based on the all-atom molecular dynamics (MD) simulation approach. A high-pressure-stimulated fluid-to-gel phase transition of the membrane is seen at 800 bar, which is consistent with previous experiments. We have also observed that in the low-pressure region (1-400 bar), urea slightly increases the membrane fluidity where TMAO decreases the same. However, the phase transition pressure remains almost unchanged on the addition of urea while TMAO shifts the phase transition toward a lower pressure. We have found that the hydrogen (H)-bond interaction between lipid and urea plays an important role in preserving the fluidity of the membrane in the low-pressure zone. However, at a higher pressure, both water and urea are excluded from the membrane surface. TMAO is also excluded from the interfacial region of the membrane at all pressures. Exclusion from the membrane surface further triggers the phase transition of the lipid membrane from the fluid to gel phase at a high pressure.

How Do Urea and Trimethylamine N-Oxide Influence the Dehydration-Induced Phase Transition of a Lipid Membrane?

Living organisms are often exposed to extreme dehydration, which is detrimental to the structure and function of the cell membrane. The lipid membrane undergoes fluid-to-gel phase transition due to dehydration and thus loses fluidity and functionality. To protect the fluid phase of the bilayer these organisms adopt several strategies. Enhanced production of small polar organic solutes (also called osmolytes) is one such strategy. Urea and trimethylamine N-oxide (TMAO) are two osmolytes found in different organisms combating osmotic stress. Previous experiments have found that both these osmolytes have strong effects on lipid membrane under different hydration conditions. Urea prevents the dehydration-induced phase transition of the lipid membrane by directly interacting with the lipids, while TMAO does not inhibit the phase transition. To provide atomistic insights, we have carried out all-atom molecular dynamics (MD) simulation of a lipid membrane under varying hydration levels and studied the effect of these osmolytes on different structural and dynamic properties of the membrane. This study suggests that urea significantly inhibits the dehydration-induced fluid-to-gel phase transition by strongly interacting with the lipid membrane via hydrogen bonds, which balances the reduced lipid hydration due to the decreasing water content. In contrast, TMAO is excluded from the membrane surface due to unfavorable interaction with the lipids. This induces further dehydration of the lipids which reinforces the fluid-to-gel phase transition. We have also studied the counteractive role of TMAO on the effect of urea on lipid membrane when both the osmolytes are present. TMAO draws some urea molecules out of the membrane and thereby reduces the effect of urea on the lipid membrane at lower hydration levels. This is similar to the counteraction of urea's deleterious effects on protein by TMAO. All these observations are consistent with the experimental results and thus provide deep molecular insights into the role of these osmolytes in protecting the fluid phase of the membrane, the key survival strategy against osmotic-stress-induced dehydration.

Effect of TMAO on the Structure and Phase Transition of Lipid Membranes: Potential Role of TMAO in Stabilizing Cell Membranes under Osmotic Stress.

Extremophiles adopt strategies to deal with different environmental stresses, some of which are severely damaging to their cell membrane. To combat high osmotic stress, deep-sea organisms synthesize osmolytes, small polar organic molecules, like trimethylamine-N-oxide (TMAO), and incorporate them in the cell. TMAO is known to protect cells from high osmotic or hydrostatic pressure. Several experimental and simulation studies have revealed the roles of such osmolytes on stabilizing proteins. In contrast, the effect of osmolytes on the lipid membrane is poorly understood and broadly debated. A recent experiment has found strong evidence of the possible role of TMAO in stabilizing lipid membranes. Using the molecular dynamics (MD) simulation technique, we have demonstrated the effect of TMAO on two saturated fully hydrated lipid membranes in their fluid and gel phases. We have captured the impact of TMAO's concentration on the membrane's structural properties along with the fluid/gel phase transition temperatures. On increasing the concentration of TMAO, we see a substantial increase in the packing density of the membrane (estimated by area, thickness, and volume) and enhancement in the orientational order of lipid molecules. Having repulsive interaction with the lipid head group, the TMAO molecules are expelled away from the membrane surface, which induces dehydration of the lipid head groups, increasing the packing density. The addition of TMAO also increases the fluid/gel phase transition temperature of the membrane. All of these results are in close agreement with the experimental observations. This study, therefore, provides a molecular-level understanding of how TMAO can influence the cell membrane of deep-sea organisms and help in combating the osmotic stress condition.