Continuous wave electron paramagnetic resonance spectroscopy and two-pulse echo detected spectra of chain-labeled lipids are used to study the dynamics of frozen lipid membranes over the temperature range 77-260 K. Bilayers of ester-linked dihexadecanoylphosphatidylcholine (DPPC) with noninterdigitated chains and ether-linked dihexadecyl phosphatidylcholine (DHPC) with interdigitated chains are considered. Rapid stochastic librations of small angular amplitude are found in both lipid matrices. In noninterdigitated DPPC bilayers, the mean-square angular amplitude, [Formula: see text], of the motion increases with temperature and it is larger close to the chain termini than close to the polar/apolar interface. In contrast, in interdigitated DHPC lamellae, [Formula: see text] is small and temperature and label-position independent at low temperature and increases steeply at high temperature. The rotational correlation time, τ, of librations lies in the subnanosecond range for DPPC and in the nanosecond range for DHPC. In all membrane samples, the temperature dependence of [Formula: see text] resembles that of the mean-square atomic displacement revealed by neutron scattering and a dynamical transition is detected in the range 210-240 K. The results highlight the librational oscillations and the glass-like behavior in bilayer and interdigitated lipid membranes.
Echo-detected EPR spectra are used to study the effects of acyl chain unsaturation on the librational motion of chain-labeled lipids in the low-temperature phases of POPC and DOPC bilayers.
Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTSs), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTSs, we extended the original set of spin-labeled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuouswave electron paramagnetic resonance spectra of spin-labeled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker length, with the longest linker placing the spin label at the entrance to the binding site. Temperature-related changes in spectral lineshapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D 2 O-electron spin echo envelope modulation in pulse-electron paramagnetic resonance measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels, implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled.
Methods of electron spin echo of pulse electron paramagnetic resonance (EPR) spectroscopy are increasingly employed to investigate biophysical properties of nitroxide-labeled biosystems at cryogenic temperatures. Two-pulse echo-detected ED-spectra have proven to be valuable tools to describe the librational dynamics in the low-temperature phases of both lipids and proteins in membranes. The motional parameter, α2τC, given by the product of the mean-square angular amplitude, α2, and the rotational correlation time, τC, of the motion, is readily determined from the nitroxide ED-spectra as well as from the W-relaxation rate curves. An independent evaluation of α2 is obtained from the motionally averaged 14N-hyperfine splitting separation in the continuous wave cw-EPR spectra. Finally, the rotational correlation time τC can be estimated by combining ED- and cw-EPR data. In this mini-review, results on the librational dynamics in model and natural membranes are illustrated.
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