Pulse EPR Detection of Lipid Exchange between Protein-Rich Raft and Bulk Domains in the Membrane: Methodology Development and Its Application to Studies of Influenza Viral Membrane

Abstract

A pulse saturation-recovery electron paramagnetic resonance (EPR) method has been developed that allows estimation of the exchange rates of a spin-labeled lipid between the bulk domain and the protein-rich membrane domain, in which the rate of collision between the spin label and molecular oxygen is reduced (slow-oxygen transport domain, or SLOT domain). It is based on the measurements of saturation-recovery signals of a lipid spin label as a function of concentrations of both molecular oxygen and the spin label. Influenza viral membrane, one of the simplest paradigms for the study of biomembranes, showed the presence of two membrane domains with slow and fast collision rates with oxygen (a 16-fold difference) at 30 degrees C. The outbound rate from and the inbound rate into the SLOT domain (or possibly the rate of the domain disintegration and formation) were estimated to be 7.7 x 10(4) and 4.6 x 10(4) s(-1), (15 micros residency time), respectively, indicating that the SLOT domain is highly dynamic and that the entire SLOT domain represents about one-third of the membrane area. Because the oxygen transport rate in the SLOT domain is a factor of two smaller than that in purple membrane, where bacteriorhodopsin is aggregated, we propose that the SLOT domain in the viral membrane is the cholesterol-rich raft domain stabilized by the trimers of hemagglutinin and/or the tetramers of neuraminidase.

Detection of Lipid Domains in Model and Cell Membranes by Fluorescence Lifetime Imaging Microscopy of Fluorescent Lipid Analogues

“…Single particle tracking experiments indicated that single glycosylphosphatidylinositolanchored proteins are organized in very small domains (Ͻ10 nm) with a short lifetime being in the order of 0.1 ms (52). This short transient stability is in agreement with electron spin resonance measurements revealing that spin-labeled lipids reside within a typical time of ϳ100 ns in rafts (53). Based on these characteristic properties of lipid domains and our estimates on the resolution of FLIM measurements (see above), our approach should be suitable to detect even nanometer-sized domains with very short stability.…”

Ca++-dependent vesicle release from erythrocytes involves stomatin-specific lipid rafts, synexin (annexin VII), and sorcin

“…58 Moreover, it has been shown that viral assembly takes place within lipid rafts of the plasma membrane and that the viral envelope is enriched in lipid raft components. [59][60][61] Our data suggest that the calcium-induced microvesiculation and nanovesiculation of erythrocytes are also lipid raft-based processes, with stomatin and synexin being the major raft proteins, respectively. It remains to be determined whether the membrane-aggregating 38 and fusogenic 62,63 activities of synexin play a role in these processes, with sorcin being a modulator.…”

The liquid-ordered phase in sphingomyelincholesterol membranes as detected by the discrimination by oxygen transport (DOT) method

“…Small rafts can sometimes be stabilized to form larger platforms through proteinprotein and protein-lipid interactions." The dynamic character of rafts has also been shown in other studies [14][15][16]. Suzuki et al [15,16] discussed the possible role of dynamic raft microdomains in signal transduction in the plasma membrane.…”