Justyna Widomska

Physical properties of lipid bilayers from EPR spin labeling and their influence on chemical reactions in a membrane environment

The influence of a variety of microenvironmental factors on the inherent reactivity of membrane-located reagents is poorly understood. A goal of this review is to provide detailed profiles of membrane properties, including hydrophobicity, oxygen and nitric oxide solubility and diffusion rates, bilayer penetration of metal ions and metal-ion complexes, and membrane order and fluidity, that can be obtained with EPR spin-labeling methods. These properties can drastically vary with membrane composition, membrane depth, and membrane domain formation, influencing the fate of chemical reactions that occur in a lipid bilayer environment.

Location of macular xanthophylls in the most vulnerable regions of photoreceptor outer-segment membranes

Lutein and zeaxanthin are two dietary carotenoids that compose the macular pigment of the primate retina. Another carotenoid, meso-zeaxanthin, is formed from lutein in the retina. A membrane location is one possible site where these dipolar, terminally dihydroxylated carotenoids, named macular xanthophylls, are accumulated in the nerve fibers and photoreceptor outer segments. Macular xanthophylls are oriented perpendicular to the membrane surface, which ensures their high solubility, stability, and significant effects on membrane properties. It was recently shown that they are selectively accumulated in membrane domains that contain unsaturated phospholipids, and thus are located in the most vulnerable regions of the membrane. This location is ideal if they are to act as lipid antioxidants, which is the most accepted mechanism through which lutein and zeaxanthin protect the retina from age-related macular degeneration. In this mini-review, we examine published data on carotenoid-membrane interactions and present our hypothesis that the specific orientation and location of macular xanthophylls maximize their protective action in membranes of the eye retina.

Functions of Cholesterol and the Cholesterol Bilayer Domain Specific to the Fiber-Cell Plasma Membrane of the Eye Lens

The most unique feature of the eye lens fiber-cell plasma membrane is its extremely high cholesterol content. Cholesterol saturates the bulk phospholipid bilayer and induces formation of immiscible cholesterol bilayer domains (CBDs) within the membrane. Our results (based on EPR spin-labeling experiments with lens-lipid membranes), along with a literature search, have allowed us to identify the significant functions of cholesterol specific to the fiber-cell plasma membrane, which are manifest through cholesterol–membrane interactions. The crucial role is played by the CBD. The presence of the CBD ensures that the surrounding phospholipid bilayer is saturated with cholesterol. The saturating cholesterol content in fiber-cell membranes keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, the CBD helps to maintain lens-membrane homeostasis when the membrane phospholipid composition changes significantly. The CBD raises the barrier for oxygen transport across the fiber-cell membrane, which should help to maintain a low oxygen concentration in the lens interior. It is hypothesized that the appearance of the CBD in the fiber-cell membrane is controlled by the phospholipid composition of the membrane. Saturation with cholesterol smoothes the phospholipid-bilayer surface, which should decrease light scattering and help to maintain lens transparency. Other functions of cholesterol include formation of hydrophobic and rigidity barriers across the bulk phospholipid-cholesterol domain and formation of hydrophobic channels in the central region of the membrane for transport of small, nonpolar molecules parallel to the membrane surface. In this review, we provide data supporting these hypotheses.

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 degrees C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.

The immiscible cholesterol bilayer domain exists as an integral part of phospholipid bilayer membranes

Electron paramagnetic resonance (EPR) spin-labeling methods were used to study the organization of cholesterol and phospholipids in membranes formed from Chol/POPS (cholesterol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine) mixtures, with mixing ratios from 0 to 3. It was confirmed using the discrimination by oxygen transport and polar relaxation agent accessibility methods that the immiscible cholesterol bilayer domain (CBD) was present in all of the suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST) in the POPS membrane. The behavior of phospholipid molecules was monitored with phospholipid analogue spin labels (n-PCs), and the behavior of cholesterol was monitored with the cholesterol analogue spin labels CSL and ASL. Results indicated that phospholipid and cholesterol mixtures can form a membrane suspension up to a mixing ratio of ~2. Additionally, EPR spectra for n-PC, ASL, and CSL indicated that both phospholipids and cholesterol exist in these suspensions in the lipid-bilayer-like structures. EPR spectral characteristics of n-PCs (spin labels located in the phospholipid cholesterol bilayer, outside the CBD) change with increase in the cholesterol content up to and beyond the CST. These results present strong evidence that the CBD forms an integral part of the phospholipid bilayer when formed from a Chol/POPS mixture up to a mixing ratio of ~2. Interestingly, CSL in cholesterol alone (without phospholipids) when suspended in buffer does not detect formation of bilayer-like structures. A broad, single-line EPR signal is given, similar to that obtained for the dry film of cholesterol before addition of the buffer. This broad, single-line signal is also observed in suspensions formed for Chol/POPS mixtures (as a background signal) when the Chol/POPS ratio is much greater than 3. It is suggested that the EPR spin-labeling approach can discriminate and characterize the fraction of cholesterol that forms the CBD within the phospholipid bilayer.

Why has Nature Chosen Lutein and Zeaxanthin to Protect the Retina

Age-related macular degeneration (AMD) is associated with a low level of macular carotenoids in the eye retina. Only two carotenoids, namely lutein and zeaxanthin are selectively accumulated in the human eye retina from blood plasma where more than twenty other carotenoids are available. The third carotenoid which is found in the human retina, meso-zeaxanthin is formed directly in the retina from lutein. All these carotenoids, named also macular xanthophylls, play key roles in eye health and retinal disease. Macular xanthophylls are thought to combat light-induced damage mediated by reactive oxygen species by absorbing the most damaging incoming wavelength of light prior to the formation of reactive oxygen species (a function expected of carotenoids in nerve fibers) and by chemically and physically quenching reactive oxygen species once they are formed (a function expected of carotenoids in photoreceptor outer segments). There are two major hypotheses about the precise location of macular xanthophylls in the nerve fiber layer of photoreceptor axons and in photoreceptor outer segments. According to the first, macular xanthophylls transversely incorporate in the lipid-bilayer portion of membranes of the human retina. According to the second, macular xanthophylls are protein-bound by membrane-associated, xanthophyll-binding proteins. In this review we indicate specific properties of macular xanthophylls that could help explain their selective accumulation in the primate retina with special attention paid to xanthophyll-membrane interactions.

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies.

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454-1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid-cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 degrees C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.

Saturation-Recovery Electron Paramagnetic Resonance Discrimination by Oxygen Transport (DOT) Method for Characterizing Membrane Domains

The discrimination by oxygen transport (DOT) method is a dual-probe saturation-recovery electron paramagnetic resonance approach in which the observable parameter is the spin-lattice relaxation time (T1) of lipid spin labels, and the measured value is the bimolecular collision rate between molecular oxygen and the nitroxide moiety of spin labels. This method has proven to be extremely sensitive to changes in the local oxygen diffusion-concentration product (around the nitroxide moiety) because of the long T1 of lipid spin labels (1-10 micros) and also because molecular oxygen is a unique probe molecule. Molecular oxygen is paramagnetic, small, and has the appropriate level of hydrophobicity that allows it to partition into various supramolecular structures such as different membrane domains. When located in two different membrane domains, the spin label alone most often cannot differentiate between these domains, giving very similar (indistinguishable) conventional electron paramagnetic resonance spectra and similar T1 values. However, even small differences in lipid packing in these domains will affect oxygen partitioning and oxygen diffusion, which can be easily detected by observing the different T1s from spin labels in these two locations in the presence of molecular oxygen. The DOT method allows one not only to distinguish between the different domains, but also to obtain the value of the oxygen diffusion-concentration product in these domains, which is a useful physical characteristic of the organization of lipids in domains. Profiles of the oxygen diffusion-concentration product (the oxygen transport parameter) in coexisting domains can be obtained in situ without the need for the physical separation of the two domains. Furthermore, under optimal conditions, the exchange rate of spin-labeled molecules between the two domains could be measured.

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

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.

Studying Lipid Organization in Biological Membranes Using Liposomes and EPR Spin Labeling

Electron paramagnetic resonance (EPR) spin-labeling methods provide a unique opportunity to determine the lateral organization of lipid bilayer membranes by discrimination of coexisting membrane domains or coexisting membrane phases. In some cases, coexisting membrane domains can be characterized without the need for their physical separation by profiles of alkyl chain order, fluidity, hydrophobicity, and oxygen diffusion-concentration product in situ. This chapter briefly explains how EPR spin-labeling methods can be used to obtain the above-mentioned profiles across lipid bilayer membranes (liposomes). These procedures will be illustrated by EPR measurements performed on multilamellar liposomes made of lipid extracts from cortical and nuclear fractions of the fiber cell plasma membrane of a cow-eye lens. To better elucidate the major factors that determine membrane properties, results for eye lens lipid membranes and simple model membranes that resemble the basic lipid composition of biological membranes will be compared.