Librational Dynamics of Spin-Labeled Membranes at Cryogenic Temperatures From Echo-Detected ED-EPR Spectra

Abstract

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, ${\alpha }^2{\tau }_C$ , given by the product of the mean-square angular amplitude, ${\alpha }^2$ , and the rotational correlation time, ${\tau }_C$ , of the motion, is readily determined from the nitroxide ED-spectra as well as from the W-relaxation rate curves. An independent evaluation of ${\alpha }^2$ is obtained from the motionally averaged ¹⁴N-hyperfine splitting separation in the continuous wave cw-EPR spectra. Finally, the rotational correlation time ${\tau }_C$ 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.

Keywords: Na, K-ATPase; echo-detected ED-spectra; electron paramagnetic resonance; electron spin echo; librations; model membranes; spin label.

FIGURE 1

(A) Two-pulse primary echo sequences and echo amplitudes decrease with increasing the interpulse delay time, τ; simulated examples of corresponding echo-detected ED-spectra of chain-labeled nitroxide in membranes. (B) Two-pulse (π/2-τ-π with microwave pulse widths of 32 and 64 ns) ED-EPR spectra of 5-PCSL in DPPC bilayers at T = 200 K recorded at incremented interpulse spacings τ (from top to bottom). Solid lines are the normalized experimental spectra, and dashed lines are simulations for isotropic librational motion. Underneath are reported the anisotropic part of the relaxation rate, W-spectra, obtained according to Eq. 1 from pairs of spectra with interpulse separations of τ ₁ = 168 and τ ₂ = 296 ns, τ ₁ = 168 ns and τ ₂ = 424 ns, or τ ₁ = 168 ns and τ ₂ = 552 ns. Schematic illustration of isotropic librational motion: the nitroxide molecule performs oscillations of small angular amplitude, α, about the three nitroxide axes. cw-EPR spectra of 5-PCSL in DPPC bilayers at 150, 220, and 260 K. ED-, W-, and cw-EPR spectra are taken from Aloi et al. (2017).

FIGURE 2

(A) Chemical structure of the lipids DPPC, DHPC, POPC, and DOPC and of the chain-labeled phosphatidylcholine spin-label 5-PCSL and 16-PCSL. Characterization of the segmental librational motion in DPPC, DHPC, POPC, and DOPC membranes spin-labeled with 5- and 16-PCSL via the temperature dependence of the (i) amplitude-correlation time product, ${\alpha }^2{\tau }_C$ , (ii) mean-square angular amplitude, ${\alpha }^2$ , and (iii) correlation time, ${\tau }_C$ . Error bars for ${\alpha }^2$ are within the symbols. Data for DPPC and DHPC are adapted from Aloi et al. (2017), and those for POPC and DOPC are from Aloi et al. (2019). (B) Na,K-ATPase membrane: crystal structure of the enzyme (PDB ID 4RES (Laursen et al., 2015)) and schematic bilayer region. Temperature dependence of the relaxation rate W _L for 5- and 14-SASL in the Na,K-ATPase membrane, in bilayers of extracted lipids and at the lipid–protein interface. Chain positional profile, that is, W _L vs. n, at T = 180 K of n-SASL in Na,K-ATPase membranes, in bilayers of the extracted lipids and at the lipid–protein interface. Temperature dependence of W _L for 14-SASL in bilayers of extracted lipids and at the lipid–protein interface and for 5-MSL in the Na,K-ATPase protein. Data are adapted from Guzzi et al. (2015).