Optimization of Transversal Relaxation of Nitroxides for Pulsed Electron−Electron Double Resonance Spectroscopy in Phospholipid Membranes

Abstract

Pulsed electron-electron double resonance (PELDOR) spectroscopy is increasingly applied to spin-labeled membrane proteins. However, after reconstitution into liposomes, spin labels often exhibit a much faster transversal relaxation (T(m)) than in detergent micelles, thus limiting application of the method in lipid bilayers. In this study, the main reasons for enhanced transversal relaxation in phospholipid membranes were investigated systematically by use of spin-labeled derivatives of stearic acid and phosphatidylcholine as well as spin-labeled derivatives of the channel-forming peptide gramicidin A under the conditions typically employed for PELDOR distance measurements. Our results clearly show that dephasing due to instantaneous diffusion that depends on dipolar interaction among electron spins is an important contributor to the fast echo decay in cases of high local concentrations of spin labels in membranes. The main difference between spin labels in detergent micelles and membranes is their local concentration. Consequently, avoiding spin clustering and suppressing instantaneous diffusion is the key step for maximizing PELDOR sensitivity in lipid membranes. Even though proton spin diffusion is an important relaxation mechanism, only in samples of low local concentrations does deuteration of acyl chains and buffer significantly prolong T(m). In these cases, values of up to 7 μs have been achieved. Furthermore, our study revealed that membrane composition and labeling position in the membrane can also affect T(m), either by promoting the segregation of spin-labeled species or by altering their exposure to matrix protons. Effects of other experimental parameters including temperature (<50 K), presence of oxygen, and cryoprotectant type are negligible under our experimental conditions.

Pulsed electron–electron double resonance (PELDOR) distance measurements in detergent micelles

“…However, utilizing the same spin-labels in phospholipid vesicle membranes homogeneous distributions have been observed. Thus, there is no indication of specific interactions [15]. Furthermore, if the spin-labels form specific structures with short spin-spin distances, their dipolar coupling will be too large to be excited by the microwave pulses.…”

“…The distribution of spin-clusters in lipid vesicle membranes can also be assumed to be homogeneous. The dimension of this homogeneous distribution varies from two to three depending on the sample concentration [13][14][15]. This corresponds to a stretched exponential decay function describing the background [16].…”

Communication: Orientational self-ordering of spin-labeled cholesterol analogs in lipid bilayers in diluted conditions

“…The effect of instantaneous diffusion was employed to measure local concentrations of free radicals appearing in irradiated solids, [10][11][12] and was observed in different other applications, including studies of spin labels in biological systems, 13,14 transition metal ions in solids, 15 phosphorus donors in silicon. 16,17 The echo decay with increasing τ is induced by also spin relaxation of electrons and nuclei, and it depends on the electron spin concentration (due to relaxation of electron spins).…”

The Topology, in Model Membranes, of the Core Peptide Derived from the T‐Cell Receptor Transmembrane Domain

“…It has been shown that a high local concentration of peptides or proteins in membranes are detrimental for DEER measurement, as it leads to a strong background decay and considerably shortens the phase memory time, T m . [35] Peptide local concentration can be reduced by decreasing the peptide/lipid ratio. It was shown for C-terminal spin-labeled gramicidin A (a membrane-embedded peptide) that increasing the molar ratio from 1:100 to 1:1000 extended T m approximately fourfold.…”

“…It was shown for C-terminal spin-labeled gramicidin A (a membrane-embedded peptide) that increasing the molar ratio from 1:100 to 1:1000 extended T m approximately fourfold. [35] In addition, by using SUVs rather than LUVs one can further reduce the local concentration by minimizing the number of peptides per vesicle. Accordingly, we chose to carry out the DEER measurements in SUVs rather than LUVs.…”