Differences in the properties of porcine cortical and nuclear fiber cell plasma membranes revealed by saturation recovery EPR spin labeling measurements.

Eye lens membranes are complex biological samples. They consist of a variety of lipids that form the lipid bilayer matrix, integral proteins embedded into the lipid bilayer, and peripheral proteins. This molecular diversity in membrane composition induces formation of lipid domains with particular physical properties that are responsible for the maintenance of proper membrane functions. These domains can be, and have been, effectively described in terms of the rotational diffusion of lipid spin labels and oxygen collision with spin labels using the saturation recovery (SR) electron paramagnetic resonance method and, now, using stretched exponential function for the analysis of SR signals. Here, we report the application of the stretched exponential function analysis of SR electron paramagnetic resonance signals coming from cholesterol analog, androstane spin label (ASL) in the lipid bilayer portion of intact fiber cell plasma membranes (IMs) isolated from the cortex and nucleus of porcine eye lenses. Further, we compare the properties of these IMs with model lens lipid membranes (LLMs) derived from the total lipids extracted from cortical and nuclear IMs. With this approach, the IM can be characterized by the continuous probability density distribution of the spin-lattice relaxation rates associated with the rotational diffusion of a spin label, and by the distribution of the oxygen transport parameter within the IM (i.e., the collision rate of molecular oxygen with the spin label). We found that the cortical and nuclear LLMs possess very different, albeit homogenous, spin lattice relaxation rates due to the rotational diffusion of ASL, indicating that the local rigidity around the spin label in nuclear LLMs is considerably greater than that in cortical LLMs. However, the oxygen transport parameter around the spin label is very similar and slightly heterogenous for LLMs from both sources. This heterogeneity was previously missed when distinct exponential analysis was used. The spin lattice relaxation rates due to either the rotational diffusion of ASL or the oxygen collision with the spin label in nuclear IMs have slower values and wider distributions compared with those of cortical IMs. From this evidence, we conclude that lipids in nuclear IMs are less fluid and more heterogeneous than those in cortical membranes. Additionally, a comparison of properties of IMs with corresponding LLMs, and lipid and protein composition analysis, allow us to conclude that the decreased lipid-to-protein ratio not only induces greater rigidity of nuclear IMs, but also creates domains with the considerably decreased and variable oxygen accessibility. The advantages and disadvantages of this method, as well as its use for the cluster analysis, are discussed.

Oxygen Transport Parameter in Plasma Membrane of Eye Lens Fiber Cells by Saturation Recovery EPR.

A probability distribution of rate constants contained within an exponential-like saturation recovery (SR) electron paramagnetic resonance signal can be constructed using stretched exponential function fitting parameters. Previously (Stein et al. Appl. Magn. Reson. 2019.), application of this method was limited to the case where only one relaxation process, namely spin-lattice relaxations due to the rotational diffusion of the spin labels in the intact eye-lens membranes, contributed to an exponential-like SR signal. These conditions were achieved for thoroughly deoxygenated samples. Here, the case is described where the second relaxation process, namely Heisenberg exchange between the spin label and molecular oxygen that occurs during bimolecular collisions, contributes to the decay of SR signals. We have further developed the theory for application of stretched exponential function to analyze SR signals involving these two processes. This new approach allows separation of stretched exponential parameters, namely characteristic stretched rates and heterogeneity parameters for both processes. Knowing these parameters allowed us to separately construct the probability distributions of spin-lattice relaxation rates determined by the rotational diffusion of spin labels and the distribution of relaxations induced strictly by collisions with molecular oxygen. The later distribution is determined by the distribution of oxygen diffusion concentration products within the membrane, which forms a sensitive new way to describe membrane fluidity and heterogeneity. This method was validated in silico and by fitting SR signals from spin-labeled intact nuclear fiber cell plasma membranes extracted from porcine eye lenses equilibrated with different fractions of air.

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.

Characterization of the distribution of spin-lattice relaxation rates of lipid spin labels in fiber cell plasma membranes of eye lenses with a stretched-exponential function.

The stretched exponential function (SEF) was used to analyze and interpret saturation recovery (SR) electron paramagnetic resonance (EPR) data obtained from spin-labeled porcine eye-lens membranes. This function has two fitting parameters: the characteristic spin-lattice relaxation rate (T 1str -1) and the stretching parameter (ß), which ranges between zero and one. When ß = 1, the function is a single exponential. It is assumed that the SEF arises from a distribution of single exponential functions, each described by a T 1 value. Because T 1 -1s are determined primarily by the rotational diffusion of spin labels, they are a measure of membrane fluidity. Since ß describes the distribution of T 1 -1s, it can be interpreted as a measure of membrane heterogeneity. The SEF was used to analyze SR data obtained from intact cortical and nuclear fiber cell plasma membranes extracted from the eye lenses of two-year old animals and spinlabeled with phospholipid- and cholesterol-analogs. The lipid environment sensed by these probe molecules was found to be less fluid and more heterogeneous in nuclear membranes than in cortical membranes. Parameters T 1str -1 and ß were also used for a multivariate K-means cluster analysis of stretched-exponential data. This analysis indicates that SEF data can be assigned accurately to clusters in nuclear or cortical membranes. In future work, the SEF will be applied to analyze data from human eye lenses of donors with differing health histories.

Spin-labeled small unilamellar vesicles with the T1-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.

Rate constants for saturation-recovery EPR and ELDOR of 14N-Spin labels.

Saturation-recovery (SR)-EPR can determine electron spin-lattice relaxation rates in liquids over a wide range of effective viscosity, making it especially useful for biophysical and biomedical applications. Here, I develop exact solutions for the SR-EPR and SR-ELDOR rate constants of 14N-nitroxyl spin labels as a function of rotational correlation time and spectrometer operating frequency. Explicit mechanisms for electron spin-lattice relaxation are: rotational modulation of the N-hyperfine and electron-Zeeman anisotropies (specifically including cross terms), spin-rotation interaction, and residual frequency-independent vibrational contributions from Raman processes and local modes. Cross relaxation from mutual electron and nuclear spin flips, and direct nitrogen nuclear spin-lattice relaxation, also must be included. Both the latter are further contributions from rotational modulation of the electron-nuclear dipolar interaction (END). All the conventional liquid-state mechanisms are defined fully by the spin-Hamiltonian parameters; only the vibrational contributions contain fitting parameters. This analysis gives a firm basis for interpreting SR (and inversion recovery) results in terms of additional, less standard mechanisms.

Multilamellar Liposomes as a Model for Biological Membranes: Saturation Recovery EPR Spin-Labeling Studies.

EPR spin labeling has been used extensively to study lipids in model membranes to understand their structures and dynamics in biological membranes. The lipid multilamellar liposomes, which are the most commonly used biological membrane model, were prepared using film deposition methods and investigated with the continuous wave EPR technique (T2-sensitive spin-labeling methods). These investigations provided knowledge about the orientation of lipids, their rotational and lateral diffusion, and their rate of flip-flop between bilayer leaflets, as well as profiles of membrane hydrophobicity, and are reviewed in many papers and book chapters. In the early 1980s, the saturation recovery EPR technique was introduced to membrane studies. Numerous T1-sensitive spin-label methods were developed to obtain detailed information about the three-dimensional dynamic membrane structure. T1-sensitive methods are advantageous over T2-sensitive methods because the T1 of spin labels (1-10 µs) is 10 to 1000 times longer than the T2, which allows for studies of membrane dynamics in a longer time-space scale. These investigations used multilamellar liposomes also prepared using the rapid solvent exchange method. Here, we review works in which saturation recovery EPR spin-labeling methods were applied to investigate the properties of multilamellar lipid liposomes, and we discuss their relationships to the properties of lipids in biological membranes.

Spin-Lattice Relaxation Rates of Lipid Spin Labels as a Measure of Their Rotational Diffusion Rates in Lipid Bilayer Membranes.

The spin-lattice relaxation rate (T1-1) of lipid spin labels obtained from saturation recovery EPR measurements in deoxygenated membranes depends primarily on the rate of the rotational diffusion of the nitroxide moiety within the lipid bilayer. It has been shown that T1-1 also can be used as a qualitative convenient measure of membrane fluidity that reflects local membrane dynamics; however, the relation between T1-1 and rotational diffusion coefficients was not provided. In this study, using data previously presented for continuous wave and saturation recovery EPR measurements of phospholipid analog spin labels, one-palmitoyl-2-(n-doxylstearoyl)phosphatidylcholine in 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine/cholesterol membranes, we show that measured T1-1 values are linear functions of rotational diffusion of spin labels. Thus, these linear relationships can be used to transfer T1-1 values into spin label rotational rates as a precise description of membrane fluidity. This linearity is independent through the wide range of conditions including lipid environment, depth in membrane, local hydrophobicity, and the anisotropy of rotational motion. Transferring the spin-lattice relaxation rates into the rotational diffusion coefficients makes the results obtained from saturation recovery EPR spin labeling easy to understand and readily comparable with other membrane fluidity data.