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

Abstract

Lipid-cholesterol interactions are responsible for different properties of biological membranes including those determining formation in the membrane of spatial inhomogeneities (lipid rafts). To get new information on these interactions, electron spin echo (ESE) spectroscopy, which is a pulsed version of electron paramagnetic resonance (EPR), was applied to study 3β-doxyl-5α-cholestane (DCh), a spin-labeled analog of cholesterol, in phospholipid bilayer consisted of equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine. DCh concentration in the bilayer was between 0.1 mol.% and 4 mol.%. For comparison, a reference system containing a spin-labeled 5-doxyl-stearic acid (5-DSA) instead of DCh was studied as well. The effects of "instantaneous diffusion" in ESE decay and in echo-detected (ED) EPR spectra were explored for both systems. The reference system showed good agreement with the theoretical prediction for the model of spin labels of randomly distributed orientations, but the DCh system demonstrated remarkably smaller effects. The results were explained by assuming that neighboring DCh molecules are oriented in a correlative way. However, this correlation does not imply the formation of clusters of cholesterol molecules, because conventional continuous wave EPR spectra did not show the typical broadening due to aggregation of spin labels and the observed ESE decay was not faster than in the reference system. So the obtained data evidence that cholesterol molecules at low concentrations in biological membranes can interact via large distances of several nanometers which results in their orientational self-ordering.

Attenuating Staphylococcus aureus Virulence by Targeting Flotillin Protein Scaffold Activity

“…ALP thus alter the plasma membrane, specifically lipid raft composition and biophysical properties (Castro et al, 2013; de Sa et al, 2015). The probe 5-DSA accumulates in biological membranes and is conventionally used to monitor membrane fluidity in studies of lipid raft organization (Grammenos et al, 2010; Kardash and Dzuba, 2014). 5-DSA can also displace certain membrane lipids and alter the function of membrane-associated proteins (Jezek et al, 1995; Wu and Gaffney, 2006).…”

Flotillin scaffold activity contributes to type VII secretion system assembly in Staphylococcus aureus

“…Simvastatin is a competitive inhibitor of HMG-CoA reductase, an enzyme upstream of squalene synthase in the constituent lipid biosynthesis pathway [ 70 ]. We included the small molecule 5-DSA (5-doxyl-stearic acid), a lipid probe that accumulates in biological membranes and is used to monitor membrane fluidity in lipid raft organization studies in eukaryotic cells [ 71 , 72 ]. In addition, 5-DSA is reported to displace certain membrane lipids and alter the function of various membrane-associated proteins [ 73 , 74 ].…”

Lipid-Mediated Clusters of Guest Molecules in Model Membranes and Their Dissolving in the Presence of Lipid Rafts

“…Our previous ESE experiments with DChl incorporated into Chol-free DOPC/DPPC bilayers have shown that DChl molecules form lipid-mediated clusters with the strong orientational correlation between the neighboring molecules. This interpretation was later confirmed by CW EPR data, which were interpreted within the model of single-crystal-like structures formed by DChl molecules in the Chol-free bilayer.…”

“…The previous ESE experiments involved extraction of the d–d contribution by comparing the ESE decays at two field positions. Here, the measurements were performed using the alternative approach, i.e., by comparing the decays for two different microwave amplitudes B 1 (see above) with the same field positionat the maximum of the EPR spectrum (position 1 in the inset to Figure ).…”

“…Pure d–d contribution from the experimental ESE signal decay was extracted using the so-called “instantaneous spectral diffusion” effect. ,− This approach has already been known for the detection of nanoscale clusters of paramagnetic centers of different origin in different media, − ,− including lipid-mediated clusters of spin-labeled molecules found recently in frozen lipid bilayers. − A brief description of the phenomenon and results of model calculations are given below.…”