Spin-labels, EPR

Strzalka, K. and W.I. Gruszecki. 1994. Effect of beta-carotene on structural and dynamic properties of model phosphatidylcholine membranes. I. An EPR spin label study. Biochim. Biophys. Acta 1194 138-142. 

EPR Spin-Labeling Demonstrates Membrane Properties Significant for Chemical... 

We would like to point out that an order parameter indicates the static property of the lipid bilayer, whereas the rotational motion, the oxygen transport parameter (Section 4.1), and the chain bending (Section 4.4) characterize membrane dynamics (membrane fluidity) that report on rotational diffusion of alkyl chains, translational diffusion of oxygen molecules, and frequency of alkyl chain bending, respectively. The EPR spin-labeling approach also makes it possible to monitor another bulk property of lipid bilayer membranes, namely local membrane hydrophobicity. 

The hypothesis that polar carotenoids regulate membrane fluidity of prokaryotes (performing a function similar to cholesterol in eukaryotes) was postulated by Rohmer et al. (1979). Thus, the effects of polar carotenoids on membrane properties should be similar in many ways to the effects caused by cholesterol. These similarities were demonstrated using different EPR spin-labeling approaches in which the effects of dipolar, terminally dihydroxylated carotenoids such as lutein,... 

Recently, due to increased interest in membrane raft domains, extensive attention has been paid to the cholesterol-dependent liquid-ordered phase in the membrane (Subczynski and Kusumi 2003). The pulse EPR spin-labeling DOT method detected two coexisting phases in the DMPC/cholesterol membranes the liquid-ordered and the liquid-disordered domains above the phase-transition temperature (Subczynski et al. 2007b). However, using the same method for DMPC/lutein (zeaxanthin) membranes, only the liquid-ordered-like phase was detected above the phase-transition temperature (Widomska, Wisniewska, and Subczynski, unpublished data). No significant differences were found in the effects of lutein and zeaxanthin on the lateral organization of lipid bilayer membranes. We can conclude that lutein and zeaxanthin—macular xanthophylls that parallel cholesterol in its function as a regulator of both membrane fluidity and hydrophobicity—cannot parallel the ability of cholesterol to induce liquid-ordered-disordered phase separation. 

EPR SPIN-LABELING DEMONSTRATES MEMBRANE PROPERTIES SIGNIFICANT FOR CHEMICAL REACTIONS AND PHYSICAL PROCESSES INVOLVING CAROTENOIDS... 

Subczynski, W. K. and A. Kusumi. 2003. Dynamics of raft molecules in the cell and artificial membranes Approaches by pulse EPR spin labeling and single molecule optical microscopy. Biochim. Biophys. Acta 1610 231-243. 

Subczynski, W. K., J. Widomska, and J. B. Feix. 2009. Physical properties of lipid bilayers from EPR spin labeling and their influence on chemical reactions in a membrane environment. Free Radic. Biol. Med., 46, 707-718. 

Widomska, J., M. Raguz, J. Dillon, E. R. Gaillard, and W. K. Subczynski. 2007. Physical properties of the lipid bilayer membrane made of calf lens lipids EPR spin labeling studies. Biochim. Biophys. Acta 1768 1454-1465. 

P3 The goal of the proposed research is to investigate the structural and dynamic properties of integral membrane proteins through the use of EPR spin-label spectroscopy and solid-state NMR spectroscopy. (From Lorigan, 2002)... 

Halestrap initially concluded that the increases in mitochondrial pyruvate transport and carboxylation were due to an increase in A pH secondary to stimulation of the electron transport chain in the cytochrome fee, region [255]. The conclusion was based largely on spectral measurements of the redox state of these cytochromes in the control and stimulated states. The spectral measurements were later found to be artifactual due to low amplitude Ca swelling of the mitochondria. Halestrap then suggested that the stable changes in the mitochondria might reside in the lipid components of the membrane due to phospholipase A 2 activity [261,262], but he has been unable to confirm this with lysophospholipid measurements [263]. On the other hand, using an EPR spin label probe of the lipid environment of the isolated mitochondria, Hoek has found differences between control and treated mitochondria [264]. 

Subczynski WK, MarkowskaE, and Sielewiesiuk J (1991) Effect of polar carotenoids on the oxygen diffusion-concentration product in lipid bilayers. An EPR spin label study. Biochim Biophys Acta 1068 68-72... 

Elsewhere in this volume Millhauser et al. have discussed the application of nitroxide electron paramagnetic resonance (EPR) spin labels to the study of the structure and dynamics of biopolymers. Another type of EPR spin label that also is useful for investigating biopolymer systems is provided by the photoexcited triplet state of an intrinsic chromophore, because a triplet state carries electronic paramagnetism. A major advantage of the photoexcited triplet state of an intrinsic chromophore over an extrinsic spin label such as a nitroxide adduct is the relatively small structural perturbation caused by the former, which consists only of a localized electronic excitation. Although not as widely exploited as fluorescence, the phosphorescence of proteins, originating from the photoexcited triplet state, has received a great deal of attention. EPR afficionados have a natural attraction to photoexcited triplet states that dates back to the... 

A similar result was obtained In a related study of frog muscle labeled with enriched glycine (46) and Chinese hamster ovary cells loaded with phosphoryl [Me- C] choline (50). These values are considerably less than that (>500 cp) obtained by Keith and Snipes (53) for chlamydomonas on the basis of an EPR spin label Study. The validity of the latter result has been questioned by Finch and Harmon (54) who suggested that the correlation time of the spin label probe could be a weighted average of Its predominant occupation of a "mobile" site and a small occupation of a highly immobilized one. 

A combination of circular dichroism, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chemical crosslinking, and analytical ultracentrifugation studies showed that both the apo- and metallated derivatives of H21(31-mer) form two-stranded a-helical coiled coils in aqueous solution. Further characterization of these derivatives by EPR spin-label experiments helped to determine its three-dimensional backbone structure. In these studies, a Cys-21 mutant of the 31-mer coiled coil, H21/C21(31-mer), was prepared and labeled with a thiol-specific nltroxide spin label (MTSL = l-oxyl-2,2,5,5-tetramethyl-A -pyrroline-3-methyl-methanethiosulfonate) at position 21 of the peptide sequence which is the site of metal substitution in the ET heterodimer. Comparison of the low-temperature, dipolar-broadened spectrum of the spin-labeled dimer with those of magnetically dilute peptide samples yielded a backbone-to-backbone distance that was nearly identical to that of the GCN4 homodimer. Based on these results, computer modeling studies provided an estimate of the metal-to-metal distance in the ET heterodimer of m-m > 25 A. The electron-transfo properties of this system are now being studied by a combination of laser flash-quench and pulse radiolysis techniques. 

The value of S obtained from the EPR data provides an analytical tool for comparison of the formulation of topical skin protectants and reactive topical skin protectants. The changes in the interior of this heterogeneous system of the labelled-TSP or la-belled-rTSP (controls) were compared with exposed labelled-TSP/or rTSP. The spin-labelled formulations were exposed to the vesicant agents in a dose/time-dependent manner. The results show that the EPR/ spin-labelling technique provides an analytical tool for determining the resistance of rTSP to the breakthrough and fluxes of vesicant agents. 

Arroyo CM and Janny SJ (1995) EPR/Spin label technique as an analytical tool for determining the resistance of reactive topical skin protectant (rTSPs) to the breakthrough of vesicant agents. Journal of Pharmacological and Toxicological Methods 33 109-112. 

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