Saturation Recovery Electron Paramagnetic Resonance Research Articles

Membrane Models and Experiments Suitable for Studies of the Cholesterol Bilayer Domains.

Cholesterol (Chol) is an essential component of animal cell membranes and is most abundant in plasma membranes (PMs) where its concentration typically ranges from 10 to 30 mol%. However, in red blood cells and Schwann cells, PMs Chol content is as high as 50 mol%, and in the PMs of the eye lens fiber cells, it can reach up to 66 mol%. Being amphiphilic, Chol molecules are easily incorporated into the lipid bilayer where they affect the membrane lateral organization and transmembrane physical properties. In the aqueous phase, Chol cannot form free bilayers by itself. However, pure Chol bilayer domains (CBDs) can form in lipid bilayer membranes with the Chol content exceeding 50 mol%. The range of Chol concentrations surpassing 50 mol% is less frequent in biological membranes and is consequently less investigated. Nevertheless, it is significant for the normal functioning of the eye lens and understanding how Chol plaques form in atherosclerosis. The most commonly used membrane models are unilamellar and multilamellar vesicles (MLVs) and supported lipid bilayers (SLBs). CBDs have been observed directly using confocal microscopy, X-ray reflectometry and saturation recovery electron paramagnetic resonance (SR EPR). Indirect evidence of CBDs has also been reported by using atomic force microscopy (AFM) and fluorescence recovery after photobleaching (FRAP) experiments. The overall goal of this review is to demonstrate the advantages and limitations of the various membrane models and experimental techniques suitable for the detection and investigation of the lateral organization, function and physical properties of CBDs.

Atomistic simulations modify interpretation of spin-label oximetry data. Part 1: intensified water-lipid interfacial resistances.

The role of membrane cholesterol in cellular function and dysfunction has been the subject of much inquiry. A few studies have suggested that cholesterol may slow oxygen diffusive transport, altering membrane physical properties and reducing oxygen permeability. The primary experimental technique used in recent years to study membrane oxygen transport is saturation-recovery electron paramagnetic resonance (EPR) oximetry, using spin-label probes targeted to specific regions of a lipid bilayer. The technique has been used, in particular, to assess the influence of cholesterol on oxygen transport and membrane permeability. The reliability of such EPR recordings at the water-lipid interface near the phospholipid headgroups has been challenged by all-atom molecular dynamics (MD) simulation data that show substantive agreement with spin-label probe measurements throughout much of the bilayer. This work uses further MD simulations, with an updated oxygen model, to determine the location of the maximum resistance to permeation and the rate-limiting barrier to oxygen permeation in 1-palmitoyl,2-oleoylphosphatidylcholine (POPC) and POPC/cholesterol bilayers at 25 and 35°C. The current simulations show a spike of resistance to permeation in the headgroup region that was not detected by EPR but was predicted in early theoretical work by Diamond and Katz. Published experimental nuclear magnetic resonance (NMR) oxygen measurements provide key validation of the MD models and indicate that the positions and relative magnitudes of the phosphatidylcholine resistance peaks are accurate. Consideration of the headgroup-region resistances predicts bilayer permeability coefficients lower than estimated in EPR studies, giving permeabilities lower than the permeability of unstirred water layers of the same thickness. Here, the permeability of POPC at 35°C is estimated to be 13 cm/s, compared with 10 cm/s for POPC/cholesterol and 118 cm/s for simulation water layers of similar thickness. The value for POPC is 12 times lower than estimated from EPR measurements, while the value for POPC/cholesterol is 5 times lower. These findings underscore the value of atomic resolution models for guiding the interpretation of experimental probe-based measurements.

Interaction of Alpha-Crystallin with Phospholipid Membranes

ABSTRACT Purpose/Aim: The amount of membrane-bound α-crystallin increases significantly with age and cataract formation, accompanied by a corresponding decline in the level of α-crystallin in the lens cytoplasm. The purpose of this research is to evaluate the binding affinity of α-crystallin to the phospholipid membranes as well as the physical properties of the membranes after α-crystallin binding. Materials and Methods: The continuous wave and saturation recovery electron paramagnetic resonance (EPR) methods were used to obtain the information about the binding affinity and the physical properties of the membrane. In this approach, the cholesterol analog spin label CSL was incorporated in the membrane and the binding of α-crystallin to the membrane was monitored by this spin label. Small uni-lamellar vesicles were prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with 1% of CSL. The measured membrane properties included the mobility parameter, fluidity, and the oxygen transport parameter. Results: The binding affinity (Ka ) of α-crystallin with the POPC membrane was estimated to be 4.9 ± 2.4 µM−1. The profiles of mobility parameter showed that mobility parameter decreased with an increase in the binding of α-crystallin. The profiles of spin-lattice relaxation rate showed that the spin-lattice relaxation rate decreased with an increase in binding. These results show that the binding of α-crystallin makes the membrane more immobilized near the head group region of the phospholipids. Furthermore, the profiles of the oxygen transport parameter indicated that the oxygen transport parameter decreased with an increase of binding, indicating the binding of α-crystallin forms a barrier for the passage of non-polar molecules which supports the barrier hypothesis. Conclusions: The binding of α-crystallin to the membrane alters the physical properties of the membranes, and this plays a significant role in modulating the integrity of the membranes. EPR techniques are useful in studying α-crystallin membrane interactions.

Binding of Alpha-Crystallin to Phospholipid Membrane: EPR Spin-Labeling Approach

The continuous wave and saturation recovery electron paramagnetic resonance (EPR) methods are powerful tools that can provide a great deal of information on the binding of alpha-crystallin with membrane. In this approach, the cholesterol analogue spin label CSL is incorporated in the membrane and the binding of alpha-crystallin to the membrane is monitored by this spin label. Small uni-lamellar vesicles were prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with 1% of CSL. The experiments were performed at varying concentrations of alpha-crystallin with fixed POPC concentration. Binding affinity (Ka) of alpha-crystallin with the membrane, as well as the physical properties of the membrane after the binding of alpha-crystallin was obtained. Physical properties include order parameter, rotational correlation time, fluidity and the oxygen transport parameter. The profiles of order parameter show the lipid order increase with increase in binding of alpha-crystallin. The profiles of rotational correlation time show that rotational correlation time increase with increase of binding. The profiles of spin-lattice relaxation rate show that spin-lattice relaxation rate decrease with increase in binding. These results show that binding of alpha-crystallin makes the membrane more immobilized near the head group region. Furthermore, the profiles of oxygen transport parameter indicates that oxygen transport parameter decrease with increase of binding, indicating the binding of alpha-crystallin forms barrier for the passage of non-polar molecules supporting the barrier hypothesis. Our results show that the binding of alpha-crystallin to membrane alters the physical properties of the membranes, and this play a significant role in modulating the integrity of the membranes.

Formation of cholesterol Bilayer Domains Precedes Formation of Cholesterol Crystals in Membranes Made of the Major Phospholipids of Human Eye Lens Fiber Cell Plasma Membranes

ABSTRACTPurpose/Aim: The goal of this study is to reveal how age-related changes in phospholipid (PL) composition in the fiber cell plasma membranes of the human eye lens affect the cholesterol (Chol) content at which Chol bilayer domains (CBDs) and Chol crystals start to form.Materials and Methods: Saturation-recovery electron paramagnetic resonance with spin-labeled cholesterol analogs and differential scanning calorimetry were used to determine the Chol contents at which CBDs and cholesterol crystals, respectively, start to form in in membranes made of the major PL constituents of the plasma membrane of the human eye lens fiber cells. To preserve compositional homogeneity throughout the membrane suspension, the lipid multilamellar dispersions investigated in this work were prepared using a rapid solvent exchange method. The cholesterol content changed from 0 to 75 mol%.Results: The saturation recovery electron paramagnetic resonance results show that CBDs start to form at 33, 50, 46, and 48 mol% Chol in the phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and sphingomyelin bilayers, respectively. The differential scanning calorimetry results show that Chol crystals start to form at 50, 66, 70, and 66 mol% Chol in the phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and sphingomyelin bilayers, respectively.Conclusions: These results, as well those of our previous studies, indicate that the formation of CBDs precedes the formation of Chol crystals in all of the studied systems, and the appearance of each depends on the type of PL forming the bilayer. These findings contribute to a better understanding of the molecular mechanisms involved in the regulation of Chol-dependent processes in eye lens fiber cell membranes.

An x-band continuous wave saturation recovery electron paramagnetic resonance spectrometer based on an arbitrary waveform generator.

An X-band (ca. 9-10 GHz) continuous wave saturation recovery spectrometer to measure electron spin-lattice relaxation (T1) was designed around an arbitrary waveform generator (AWG). The AWG is the microwave source and is used for timing of microwave pulses, generation of control signals, and digitizer triggering. Use of the AWG substantially simplifies the hardware in the bridge relative to that in conventional spectrometers and decreases the footprint. The bridge includes selectable paths with different power amplifications to permit experiments requiring hundreds of milliwatts to fractions of nanowatts for the pump and observe periods. The signal is detected with either a single or quadrature-output double balanced mixer. The system can operate with reflection or crossed-loop resonators. The source noise from the AWG was decreased by addition of a Wenzel high-stability clock. The source is sufficiently stable that automatic frequency control is not needed. The spectrometer was tested with samples that contained 1 × 1015 to 8 × 1017 spins and have T1 between a few hundred ns and hundreds of μs. Excellent signal-to-noise ratio was obtained with acquisition times of 2-90 s. Signal-to-noise performance is similar to that of a conventional saturation recovery spectrometer with a solid-state source. The stability and data reproducibility are better than with conventional sources. With replacement of frequency-sensitive components, this spectrometer can be used to perform saturation recovery measurements at any frequency within the range of the AWG.

Detection of cholesterol bilayer domains in intact biological membranes: Methodology development and its application to studies of eye lens fiber cell plasma membranes

Four purported lipid domains are expected in plasma membranes of the eye lens fiber cells. Three of these domains, namely, bulk, boundary, and trapped lipids, have been detected. The cholesterol bilayer domain (CBD), which has been detected in lens lipid membranes prepared from the total lipids extracted from fiber cell plasma membranes, has not yet been detected in intact fiber cell plasma membranes. Here, a saturation-recovery electron paramagnetic resonance spin-labeling method has been developed that allows identification of CBDs in intact fiber cell plasma membranes of eye lenses. This method is based on saturation-recovery signal measurements of the cholesterol-analog spin label located in the lipid bilayer portion of intact fiber cell membranes as a function of the partial pressure of molecular oxygen with which the samples are equilibrated. The capabilities and limitations of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses where CBDs were detected in porcine nuclear intact membranes for which CBDs were also detected in lens lipid membranes. CBDs were not detected in porcine cortical intact and lens lipid membranes. CBDs were detected in intact membranes isolated from both cortical and nuclear fiber cells of lenses obtained from human donors. The cholesterol content in fiber cell membranes of these donors was always high enough to induce the formation of CBDs in cortical as well as nuclear lens lipid membranes. The results obtained for intact membranes, when combined with those obtained for lens lipid membranes, advance our understanding of the role of high cholesterol content and CBDs in lens biology, aging, and/or cataract formation.

Effect of Phosphorylation on Interactions between Transmembrane Domains of SERCA and Phospholamban

We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to map interactions between the transmembrane (TM) domains of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLB) as affected by PLB phosphorylation. In the cardiac sarcoplasmic reticulum, PLB binding to SERCA results in Ca-dependent enzyme inhibition, which is reversed by PLB phosphorylation at Ser16. Previous spectroscopic studies on SERCA-PLB have largely focused on the cytoplasmic domain of PLB, showing that phosphorylation induces a structural shift in this domain relative to SERCA. However, SERCA inhibition is due entirely to TM domain interactions. Therefore, we focus here on PLB’s TM domain, attaching Cys-reactive spin labels at five different positions. In each case, continuous-wave EPR indicated moderate spin-label mobility, with the addition of SERCA revealing two populations, one indistinguishable from PLB alone and another with more restricted rotational mobility, presumably due to SERCA-binding. Phosphorylation had no effect on the rotational mobility of either component but significantly decreased the mole fraction of the restricted component. Solvent-accessibility experiments using power-saturation EPR and saturation-recovery EPR confirmed that these two spectral components were SERCA-bound and unbound PLB and showed that phosphorylation increased the overall lipid accessibility of the TM domain by increasing the fraction of unbound PLB. However—based on these results—at physiological levels of SERCA and PLB, most SERCA would have bound PLB even after phosphorylation. Additionally, no structural shift in the TM domain of SERCA-bound PLB was detected, as there were no significant changes in membrane insertion depth or its accessibility. Therefore, we conclude that under physiological conditions, the phosphorylation of PLB induces little or no change in the interaction of the TM domain with SERCA, so relief of inhibition is predominantly due to the previously observed structural shift in the cytoplasmic domain.

Saturation recovery EPR spin-labeling method for quantification of lipids in biological membrane domains.

The presence of integral membrane proteins induces the formation of distinct domains in the lipid bilayer portion of biological membranes. Qualitative application of both continuous wave (CW) and saturation recovery (SR) electron paramagnetic resonance (EPR) spin-labeling methods allowed discrimination of the bulk, boundary, and trapped lipid domains. A recently developed method, which is based on the CW EPR spectra of phospholipid (PL) and cholesterol (Chol) analog spin labels, allows evaluation of the relative amount of PLs (% of total PLs) in the boundary plus trapped lipid domain and the relative amount of Chol (% of total Chol) in the trapped lipid domain [M. Raguz, L. Mainali, W. J. O'Brien, and W. K. Subczynski (2015), Exp. Eye Res., 140:179-186]. Here, a new method is presented that, based on SR EPR spin-labeling, allows quantitative evaluation of the relative amounts of PLs and Chol in the trapped lipid domain of intact membranes. This new method complements the existing one, allowing acquisition of more detailed information about the distribution of lipids between domains in intact membranes. The methodological transition of the SR EPR spin-labeling approach from qualitative to quantitative is demonstrated. The abilities of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses. Statistical analysis (Student's t-test) of the data allowed determination of the separations of mean values above which differences can be treated as statistically significant (P ≤ 0.05) and can be attributed to sources other than preparation/technique.

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

The presence of integral membrane proteins in biological membranes induces the formation of distinct domains in the lipid bilayer portion of these membranes. We were able to discriminate the existence of three lipid environments in intact fiber cell plasma membranes of the eye lens, namely bulk, boundary, and trapped lipids. However, our qualitative approach did not allow quantification of lipids in each domain. Recently, based on the continuous wave (CW) electron paramagnetic resonance (EPR) spectra of phospholipid analog and cholesterol analog spin labels, we developed a method that allows evaluation of the relative amount of phospholipids and cholesterol in the bulk lipid domain and in the boundary plus trapped lipid domain [M. Raguz, L. Mainali,W. J. O’Brien and W. K. Subczynski, (2015), Exp. Eye Res., 140:179-186]. Here, we will present a new approach that, based on saturation-recovery (SR) EPR, allows evaluation of the relative amount of phospholipids and cholesterol in the bulk plus boundary lipid domain and in the trapped lipid domain. Spectrometer conditions for the successful application of this method to quantify lipids in membrane domains are described. These two methods clearly demonstrate that the method's time window allows the separation of results from the different domains. CW EPR mixes results from the boundary and trapped lipids, while SR EPR mixes results from the bulk and boundary lipids. These two methods complement each other, providing a more complete picture of lipid lateral organization in intact membranes. The abilities of these methods are illustrated in the intact fiber cell plasma membranes from porcine eye lenses.Acknowledgments: This work was supported by grants EY015526, EB001980, and EY001931 from the National Institutes of Health.

Oxidation of Polyunsaturated Phospholipid Decreases the Cholesterol Content at which Cholesterol Bilayer Domains Start to form in Phospholipid-Cholesterol Membranes

Saturation-recovery electron paramagnetic resonance and differential scanning calorimetry were used to determine the cholesterol content at which pure cholesterol bilayer domains (CBDs) and cholesterol crystals begin to form in 1-palmitoyl-2-arachidonoylphosphochatidyline (PAPC) membranes. To preserve compositional homogeneity of the membrane suspension at a high cholesterol content, multilamellar liposome suspensions were prepared using the rapid solvent exchange method. The cholesterol content in membranes was changed from 0 mol% to 66 mol%.

Photoaging of retinal pigment epithelial melanosomes: The effect of photobleaching on morphology and reactivity of the pigment granules

To elucidate the mechanism of age-related changes in antioxidant and photoprotective properties of human retinal pigment epithelium (RPE) melanosomes, the effect of in vitro photoaging of bovine RPE melanosomes was examined employing an array of complementary spectroscopic and analytical methods. Electron paramagnetic resonance (EPR) spectroscopy, saturation recovery EPR, atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to determine melanin content of control and photobleached melanosomes, and to monitor changes in their morphology. Methylene blue (MB), TEMPO choline, dysprosium(III) ions and singlet oxygen were employed as molecular probes to characterize the efficiency of control and photobleached melanosomes to interact with different reagents. EPR oximetry, UV–vis absorption spectroscopy, iodometric assay of lipid hydroperoxides and time-resolved singlet oxygen phosphorescence were used to analyze the efficiency of photobleached and untreated melanosomes to inhibit MB-photosensitized oxidation of liposomal lipids. The obtained results revealed that, compared to untreated melanosomes, moderately photobleached melanosomes protected unsaturated lipids less efficiently against photosensitized peroxidiation, while weakly photobleached melanosomes were actually better antioxidant and photoprotective agents. The observed changes could be attributed to two effects – modification of the melanosome morphology and oxidative degradation of the melanin functional groups induced by different degree of photobleaching. While the former increases the accessibility of melanin nanoaggregates to reagents, the latter reduces the efficiency of melanin to interact with chemical and physical agents.

Oxidation of Cholesterol and Formation of Cholesterol Hydroperoxides Decreases the Cholesterol Concentration at which Formation of Cholesterol Bilayer Domains and Cholesterol Crystals is Initiated in Phospholipid Bilayers

Recently we demonstrated that formation of cholesterol bilayer domains (CBDs) precedes formation of cholesterol crystals (CHCs) in cholesterol/dimyristoylphosphatidylcholine (Chol/DMPC) membranes (Mainali et. al., J. Phys. Chem. B, 117(2013)8994-9003). In the presented studies, we show that the oxidation of Chol in Chol/DMPC membranes induces formation of CBDs and CHCs at significantly lower Chol concentrations than in membranes without oxidation. To preserve compositional homogeneity throughout the membrane suspension we prepared the multilamellar liposomes with a rapid solvent exchange method. The oxidation of Chol was induced using a photosensitizer, rose Bengal, and the extent of oxidation (formation of Chol hydroperoxides) was measured using iodometric assay. To avoid formation of phospholipid hydroperoxides, which would interfere with our assay, the saturated phospholipid (DMPC) was used to prepare liposomes. Saturation recovery electron paramagnetic resonance with spin-labeled cholesterol analogues (androstane and cholestane spin labels) was used to detect the formation of CBDs and differential scanning calorimetry was used to detect the formation of CHCs. We conclude that formation of Chol hydroperoxides induces the formation of CBDs at Chol concentrations where previously CBDs were not present in non-oxidized membranes as well as the collapse of the already formed CBDs to CHCs, presumably outside the lipid bilayer.

Spin-Labeled Small Unilamellar Vesicles with the T 1-Sensitive Saturation-Recovery EPR Display as an Oxygen-Sensitive Analyte for Measurement of Cellular Respiration

This study validated the use of small unilamellar vesicles (SUVs) made of 1-palmitoyl-2-oleoylphosphatidylcholine with 1 mol% spin label of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine (16-PC) as an oxygen sensitive analyte to study cellular respiration. In the analyte the hydrocarbon environment surrounds the nitroxide moiety of 16-PC. This ensures high oxygen concentration and oxygen diffusion at the location of the nitroxide as well as isolation of the nitroxide moiety from cellular reductants and paramagnetic ions that might interfere with spin-label oximetry measurements. The saturation-recovery EPR approach was applied in the analysis since this approach is the most direct method to carry out oximetric studies. It was shown that this display (spin-lattice relaxation rate) is linear in oxygen partial pressure up to 100% air (159 mmHg). Experiments using a neuronal cell line in suspension were carried out at X-band for closed chamber geometry. Oxygen consumption rates showed a linear dependence on the number of cells. Other significant benefits of the analyte are: the fast effective rotational diffusion and slow translational diffusion of the spin-probe is favorable for the measurements, and there is no cross reactivity between oxygen and paramagnetic ions in the lipid bilayer.

Saturation-Recovery EPR with Nitroxyl Radical–Dy(III) Spin Pairs: Distances and Orientations

We describe a method for measuring the distance between a radical and a Dy(III) ion using saturation-recovery electron paramagnetic resonance (EPR) and demonstrate its application using four chemically modified DNA duplexes. The four DNA duplexes contain a terminal nitroxide spin-label and a midsequence, EDTA-bound Dy(III) ion but differ in the nitroxyl radical (NO)-Dy(III) distance. Distances can be determined with high precision because of their sixth-root dependence on the experimentally determined dipolar rate constant. Furthermore, the orientation of the NO-Dy(III) interspin vector in the Dy(III) g-tensor reference frame can be determined for two of the DNA duplexes. The shortest mean NO-Dy(III) distance, 18.3 ± 0.3 Å, and the longest, 50.3 ± 2.4 Å, are near the lower and upper distance limits of what can be measured with the NO-EDTA(Dy(III)) pair at X-band. These methods are applicable to structural studies of nucleic acids, proteins, and their complexes.

A New Phase Boundary in Phosphatidylcholine/Cholesterol Bilayers (In the Dimyristoylphosphatidylcholine/Cholesterol Bilayer): EPR and DSC Studies

A new phase boundary, at cholesterol/dimyristoylphosphatidylcholine (Chol/DMPC) mixing ratio of ∼1, was observed by saturation-recovery electron paramagnetic resonance (EPR) spin-labeling method in a multilamellar suspension of DMPC and Chol prepared using a rapid solvent exchange method. With spin-labeled cholesterol analogues (androstane spin label [ASL] and cholestane spin label [CSL]) it was shown that the upper limit of the cholesterol concentration in the liquid-ordered phase of the DMPC membrane is ∼50 mol%, above which the excess of cholesterol forms the pure cholesterol bilayer domain (CBD). Thus, the phase boundary at a Chol/DMPC mixing ratio of ∼1 separates the region with a single liquid-ordered phase from the region with a coexisting liquid-ordered phase and the CBD. Because the pure cholesterol cannot form the free bilayer in the buffer, the CBD has to be supported by the surrounding phospholipid bilayer saturated with cholesterol. The next phase boundary is defined by the cholesterol solubility threshold (CST) that indicates the amount of cholesterol which can saturate the DMPC bilayer and form the CBD. The excess of cholesterol above the CST forms monohydrate cholesterol crystals which precipitate outside of the lipid bilayer. It was shown by differential scanning calorimetry (DSC) that the CST, which separates the region with a coexisting liquid-ordered phase saturated with cholesterol and the CBD from the region in which cholesterol crystals are formed, is located at the Chol/DMPC mixing ratio of ∼2 (∼66 mol% cholesterol). The extended phase diagram for the DMPC/cholesterol membrane, covering the region where the membrane is saturated and oversaturated with cholesterol, is proposed.

Probing Protein Secondary Structure using EPR: Investigating a Dynamic Region of Visual Arrestin.

One key application of site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is the determination of sequence-specific secondary structure in proteins. Regular secondary structure leads to a periodic variation in both side chain motion and solvent accessibility, two properties easily monitored by EPR techniques. Specifically, saturation recovery (SR) EPR spectroscopy has proven to be useful for making accessibility measurements for multiple protein structure populations by determining individual accessibilities and is therefore well suited to study the structure of proteins exhibiting multiple conformations in equilibrium. Here we employ both continuous wave and SR EPR spectroscopy in combination to examine the secondary structure of a short sequence showing conformational heterogeneity in visual rod arrestin. The EPR data presented here clearly distinguish between the unstructured loop and the helical structure formed in the crystallographic tetramer of visual arrestin and show that this region is unstructured in solution.

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.

Phase-Separation and Domain-Formation in Cholesterol-Sphingomyelin Mixture: Pulse-EPR Oxygen Probing

Membranes made of Chol/ESM (cholesterol/egg sphingomyelin) mixtures were investigated using saturation-recovery electron paramagnetic resonance spin-labeling methods, in which bimolecular collisions of relaxation agents (oxygen or nickel ethylenediamine diacetic acid) with spin labels are measured. Liquid-disordered (ld) and liquid-ordered (lo) phases, and cholesterol bilayer domains (CBDs) were discriminated and characterized by profiles of the oxygen transport parameter (OTP). In the ld phase, coexisting with the lo phase, the OTP profile is bell-shaped and lies above that in the pure ESM membrane. Changes in the OTP profile across the lo phase are complex. When the lo phase coexists with the ld phase, the OTP profile is similar to that across the pure ESM membrane but with a steeper bell shape. With an increase in cholesterol concentration (up to the cholesterol-solubility threshold), the profile becomes rectangular, with low OTP values from the membrane surface to the depth of C9, and high values in the membrane center. This approximately threefold increase in the OTP occurs at the depth at which the rigid ring structure of cholesterol is immersed. Further addition of cholesterol and the formation of the CBD does not affect the OTP profile across the lo phase. OTP values in the CBD are significantly lower than in the lo phase.

Reorientation of cytochrome c2 upon interaction with oppositely charged macromolecules probed by SR EPR: implications for the role of dipole moment to facilitate collisions in proper configuration for electron transfer

The reaction of water-soluble cytochrome c (c(2)) with its physiological redox partners is facilitated by electrostatic attractions between the two protein surfaces. Using spin-labeled cytochrome c(2) from Rhodobacter capsulatus and pulse electron paramagnetic resonance (EPR) measurements we compared spatial orientation of cytochrome c(2) upon its binding to surfaces of opposite charge. We observed that cytochrome c(2) can use its negatively charged "back" side when exposed to interact with positively charged surfaces (DEAE resin) which is the opposite to the use of its positively charged "front" side in physiological interaction with negatively charged binding domain of cytochrome bc(1). The later orientation is also adopted upon non-physiological binding of cytochrome c(2) to negatively charged carboxymethyl cellulose resin. These results directly demonstrate how the electric dipolar nature of cytochrome c(2) influences its orientation in interactions with charged surfaces, which may facilitate collisions with other redox proteins in a proper orientation to support physiologically-competent electron transfer. Saturation recovery EPR provides an attractive tool for monitoring spatial orientation of proteins in their interaction with surfaces in liquid phase. It is particularly valuable for metalloproteins engaged in redox reactions as a means to monitor the geometry and dynamics of formation of protein complexes in measurements that are independent of electron transfer processes.