Low-Temperature Molecular Motions in Phospholipid Bilayers in Presence of Glycerol as Studied by Spin-Echo EPR of Spin Labels

Abstract

Glycerol is used as a cryoprotective agent to protect biological systems under freezing conditions. Electron spin echo (ESE) spectroscopy, a pulsed version of EPR, is capable of studying low-temperature molecular motions of nitroxide spin labels. ESE technique was applied to study molecular motions in phospholipid bilayers prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with added spin-labeled lipids 1-palmitoyl-2-stearoyl-(n-DOXYL)-sn-glycero-3-phosphocholine (n-PCSL, n was optionally 5 or 16). Bilayers were hydrated (solvated) either in pure water or in a 1:1 v/v water–glycerol mixture. In the used ESE approach, there were studied stochastic (or diffusive) orientational vibrations of the molecule as a whole (i.e., stochastic molecular librations). The anisotropic contribution to the echo decay rate, W anis, was measured, which is proportional, according to theory, to the product of the mean-squared angular amplitude \(\langle \alpha^{ 2} \rangle\) and the correlation time τ c. W anis was found to be small below and to sharply increase above 200 K, for the both types of solvents and the both label positions. As compared with hydration by pure water, in presence of glycerol W anis was larger for the 5th label position while for the 16th one it did not change. Also, for the 5th label position W anis values were found to be nearly the same as those for a polar spin probe 3,4-dicarboxy-PROXYL which was separately added to the bilayer as a reference and which is assumed to be partitioned only into the solvating shell. These results indicate that motions at the surface of bilayer are governed by the motion of solvating shell while motions in the bilayer interior occur independently. The relation of the obtained data with the dynamical transition phenomenon that is known for biological substances near 200 K from neutron scattering and Mossbauer absorption is discussed.