Membrane-Sugar Interactions Probed by Pulsed Electron Paramagnetic Resonance of Spin Labels.

Sugars can stabilize biological systems under extreme desiccation and freezing conditions. Hypothetical molecular mechanisms suggest that the stabilization effect may be determined either by specific interactions of sugars with biological molecules or by the influence of sugars on the solvating shell of the biomolecule. To explore membrane-sugar interactions, we applied electron spin echo envelope modulation (ESEEM) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR), to phospholipid bilayers with spin-labeled lipids added and solvated by aqueous deuterated sucrose and trehalose solutions. The phospholipids were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The spin-labeled lipids were 1,2-dipalmitoyl-sn-glycero-3-phospho(TEMPO)choline (T-PCSL), with spin-label TEMPO at the lipid polar headgroup. The deuterium ESEEM amplitude was calibrated using known concentrations of glassy deuterated sugar solvents. The data obtained indicated that the sugar concentration near the membrane surface obeyed a simple Langmuir model of monolayer adsorption, which assumes direct sugar-molecule bonding to the bilayer surface.

Low-temperature molecular motions in lipid bilayers in the presence of sugars: insights into cryoprotective mechanisms.

Sugars and sugar alcohols can stabilize biological systems under extreme conditions of desiccation and freezing. Phospholipid bilayers solvated by aqueous solutions of sucrose, trehalose, and sorbitol at concentrations of 0.2 and 1 M and containing incorporated spin-labeled stearic acids were studied by electron spin echo (ESE) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR). The phospholipids were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and the stearic acids were labeled with nitroxide 4,4-dimethyl-oxazolidine-1-oxyl (DOXYL) attached rigidly at either the 5th or 16th carbon positions. The ratio of the echo time traces for the two field positions in the EPR spectrum possessing the largest and smallest anisotropies gave the anisotropic contribution to the echo decay, which obeys exponential time dependence with good accuracy. At low temperatures, the anisotropic contribution is induced by stochastic (or diffusive) orientational vibrations of the molecule as a whole (i.e., stochastic molecular librations), with the exponential decay rate Wanis proportional to <α(2)>τc, where <α(2)> is the mean angular amplitude of the motion and τc is the correlation time. In all cases, it was found that Wanis begins to increase sharply above 170-200 K, which was ascribed to the dynamical transition known for biological systems at these temperatures. For hydration by the sucrose and trehalose solutions, Wanis was found to increase noticeably also above ∼120 K, which was explained by bilayer expansion due to direct bonding of sugar molecules to the bilayer surface. The Wanis temperature dependencies were found to be close to those obtained for the simple systems of the nitroxide spin probe TEMPONE in aqueous sorbitol and sugar 1 M solutions. This correlation suggests a possible mechanism of cryoprotective action of sorbitol and sugars due to the similarity of low-temperature motions in the membrane and in the cryoprotectant-containing surrounding liquid.