Using spin-label electron paramagnetic resonance (EPR) to discriminate and characterize the cholesterol bilayer domain

Abstract

Electron paramagnetic resonance (EPR) spin-labeling methods make it possible not only to discriminate the cholesterol bilayer domain (CBD) but also to obtain information about the organization and dynamics of cholesterol molecules in the CBD. The abilities of spin-label EPR were demonstrated for Chol/POPC (cholesterol/1-palmitoyl-2-oleoylphosphatidylcholine) membranes, with a Chol/POPC mixing ratio that changed from 0 to 3. Using the saturation-recovery (SR) EPR approach with cholesterol analogue spin labels, ASL and CSL, and oxygen or NiEDDA relaxation agents, it was confirmed that the CBD was present in all membrane suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST). Conventional EPR spectra of ASL and CSL in the CBD were similar to those in the surrounding POPC bilayer (which is saturated with cholesterol), indicating that in both domains, cholesterol exists in the lipid-bilayer-like structures. The behavior of ASL and CSL (and, thus, the behavior of cholesterol molecules) in the CBD was compared with that in the surrounding POPC-cholesterol domain (PCD). In the CBD, ASL and CSL molecules are better ordered than in the surrounding PCD. This difference is small and can be compared to that induced in the surrounding domain by an ~10°C decrease in temperature. Thus, cholesterol molecules are unexpectedly dynamic in the CBD, which should enhance their interaction with the surrounding domain. The polarity of the water/membrane interface of the CBD is significantly greater than that of the surrounding PCD, which significantly enhances penetration of the water-soluble relaxation agent, NiEDDA, into that region. Hydrophobicity measured in the centers of both domains is similar. The oxygen transport parameter (oxygen diffusion-concentration product) measured in the center of the CBD is about ten times smaller than that measured in the center of the surrounding domain. Thus, the CBD can form a significant barrier to oxygen transport. The results presented here point out similarities between the organization and dynamics of cholesterol molecules in the CBD and in the surrounding PCD, as well as significant differences between CBDs and cholesterol crystals.

Saturation Recovery EPR Spin-Labeling Method for Quantification of Lipids in Biological Membrane Domains

“…The exchange rate with other domains is of the order of magnitude of 10 5 s −1 or less [16]. The CBD is a highly fluid pure Chol bilayer immersed in the bulk lipids [14, 32, 33]. Figure 1 in Ref.…”

“…Because Chol molecules (and Chol-analog spin labels) are substantially excluded from the boundary lipids [22, 23], this domain would not be considered as a place where ASL (and Chol molecules) would be located. The spin-lattice relaxation time alone also cannot discriminate between the bulk domain and the CBD because the fluidity of these domains (dynamics of Chol molecules [14, 33]) is similar, giving similar T 1 values of ASL. Thus, the SR EPR approach with ASL can discriminate only the rapid exchange bulk/CBD lipid domain from the trapped lipid domain.…”

“…Problems with discriminating the CBD can be related to the fact that properties of Chol-analog spin labels in the CBD are similar to those of Chol-analog spin labels in the surrounding PL bilayer. (See [14] for additional detail. )…”

Properties of membranes derived from the total lipids extracted from clear and cataractous lenses of 61–70-year-old human donors

“…The CBD is a pure Chol domain formed within the PCD (the phospholipid bilayer saturated with Chol) and, for this reason, can be discriminated only with Chol analog spin labels ASL and CSL (Raguz et al 2011a, b). When located in these two membrane domains, these spin labels alone cannot differentiate between domains, giving similar conventional EPR spectra and similar T 1 values (Raguz et al 2011a, b).…”

“…For measurements of the NiEDDA accessibility parameter, 20 mM NiEDDA was present in the buffer, and saturation-recovery measurements were performed for deoxygenated samples (Raguz et. al 2011a, b). The measured value is the rate of bimolecular collisions between the water-soluble relaxation agent NiEDDA and the nitroxide group of the spin labels.…”