Double Electron–Electron Resonance of Spin-Labeled Cholestane in Model Membranes: Evidence for Substructures inside the Lipid Rafts

Abstract

Plasma membranes are assumed to be highly compartmentalized, which is believed to be important for the membrane protein functionality. The liquid ordered-disordered phase segregation in the membranes results in nanoscale liquid-ordered assemblieslipid rafts. Double electron–electron resonance spectroscopy (DEER, also known as PELDOR) is sensitive to spin–spin dipolar interactions between spin labels at the nanoscale range of distances. Here, DEER is applied to spin-labeled cholestane, 3β-doxyl-5α-cholestane (DChl), diluted in bilayers composed of an equimolar mixture of dioleoyl-glycero-phosphocholine (DOPC) and dipalmitoyl-glycero-phosphocholine (DPPC) phospholipids, with cholesterol (Chol) added. The DEER data allowed us to detect clustering of the DChl molecules. Their lateral distribution in the clusters in the absence of Chol was found to be random, while in the presence of Chol it became quasi-regular. DEER time traces are fairly well simulated within a simple square superlattice model. For the 20 mol % Chol content, for which at physiological temperatures, the lipid rafts are formed, the found superlattice parameter was 3.7 nm. Assuming that lipid rafts are captioned upon shock freezing at the temperature of investigation (80 K), the found regularity of DChl lateral distribution was interpreted by raft substructuring, with the DChl molecules embedded between the substructures.

Clustering of Stearic Acids in Model Phospholipid Membranes Revealed by Double Electron–Electron Resonance

“…One of the possible ways to explain these peculiarities of the DEER data for 5-DSA could be given assuming that DSA molecules form alternative subclusters in two opposite leaflets, with repulsion between DSA polar heads, like it is schematically presented in Figure . The alternation will result in nonlinearity of DEER decays, as it was shown in simulations for the regularly located spin labels . Also, the repulsion will diminish the efficient local concentration.…”

“…For a random distribution within a plane, ρ loc is replaced by σ loc that is the local surface spin concentration taken in cm –2 units, and eq is transformed into the dependence of the form of exp­( – constσ loc t 2/3 ) . Numerical calculations taking into account all plane orientations with respect to the external magnetic field result in the following approximating formula: Note also that in biological membranes, other kinds of spatial distributions may appear, e.g., it could be a regular lateral disposition of spin-labeled molecules …”

“…Note also that in biological membranes, other kinds of spatial distributions may appear, e.g., it could be a regular lateral disposition of spin-labeled molecules. 36 ■ RESULTS AND DISCUSSION DOPC/DPPC Bilayers. All CW EPR spectra for DSAs taken at 200 K and below were typical for immobilized nitroxides.…”

“…The alternation will result in nonlinearity of DEER decays, as it was shown in simulations for the regularly located spin labels. 36 Also, the repulsion will diminish the efficient local concentration. To confirm this hypothesis, further studies and theoretical modeling however are needed.…”

Synthesis of Spin-Labeled Ibuprofen and Its Interaction with Lipid Membranes

“…Spin-label EPR spectroscopy [29] allows one to obtain structural and dynamical information at the molecular level, of the kind that is accessible only with this technique. In particular, a pulsed EPR version of electron spin echo envelope modulation (ESEEM) spectroscopy probes the position of molecules in the membranes [30][31][32][33], while double electron-electron resonance (DEER, also known as PELDOR) spectroscopy probes molecular self-assembling in membranes and other biological systems [34][35][36][37][38].…”