Spin-label Spectra Research Articles

Background removal from rapid-scan EPR spectra of nitroxide-based spin labels by minimizing non-quadratic cost functions

Rapid-scan electron paramagnetic resonance spectroscopy is an emerging technique which substantially improves the signal-to-noise ratio and time resolution compared to conventional continuous-wave experiments. This allows the investigation of spin-labeled biomolecules and their structural dynamics on much shorter time scales than usually accessible. The EPR spectrum however is superimposed by a strong background that is caused by microphonic effects of the alternating magnetic field. This article discusses the use of non-quadratic cost functions for background removal of rapid-scan spectra. The method is validated for the most prominent type of spin-probes in the field of biochemistry: the nitroxide spin-label.

Regarding the measurement of microviscosity in lipid bilayers by EPR

Simulations of electron paramagnetic resonance spectra of stearic acid spin labels in lipid bilayers are used to illustrate the fact that the apparent order parameter Sapp calculated from the spectral shape does not coincide with the true order parameter S in the case of slow motions. While S reflects a static property as the degree of order of the lipid chains, Sapp depends on both order and dynamics. Thus, calibration procedures intended to obtain bilayer microviscosity values from Sapp in the slow motion regime are not reliable. However, Sapp is a useful tool to describe trends in membrane fluidity.

Ether-linked lipids: Spin-label EPR and spin echoes

Electron spin echo envelope modulation (ESEEM) and conventional electron paramagnetic resonance (EPR) of site-specifically spin-labelled phospholipids are used to investigate the effect of ether-linked chains on the water-penetration and polarity profiles, as well as the phase behaviour and chain flexibility profiles, of phospholipid membranes. D2O-ESEEM reveals that water exposure of the terminal methyl groups in the interdigitated phase of dihexadecyl phosphatidylcholine (DHPC) is comparable to that of the methylene groups at the polar head-group end of the chains. Similarly, an uniform transmembrane polarity profile is obtained from the dependence of the outer 14N-hyperfine splitting on the spin-label position along the chain in frozen interdigitated DHPC dispersions. Two-component conventional EPR spectra of spin labels at the terminal methyl end of the chain reveal that the intermediate gel phase above the pretransition of DHPC contains components in which the lipid chains are interdigitated. The polarity and chain-flexibility profiles in the fluid Lα-phase of DHPC with ether-linked chains are shifted outwards, towards the polar-apolar interface, as compared with that of dihexadecanoyl phosphatidylcholine (DPPC) with ester-linked chains. Also, the polarity profile of DHPC is shifted upwards, to higher polarities. These differences reflect those in hydrocarbon thickness and area/lipid molecule reported by x-ray diffraction for the Lα-phases of the two lipids.

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.

EPR spin labeling measurements of thylakoid membrane fluidity during barley leaf senescence

Physical properties of thylakoid membranes isolated from barley were investigated by the electron paramagnetic resonance (EPR) spin labeling technique. EPR spectra of stearic acid spin labels 5-SASL and 16-SASL were measured as a function of temperature in secondary barley leaves during natural and dark-induced senescence. Oxygen transport parameter was determined from the power saturation curves of the spin labels obtained in the presence and absence of molecular oxygen at 25°C. Parameters of EPR spectra of both spin labels showed an increase in the thylakoid membrane fluidity during senescence, in the headgroup area of the membrane, as well as in its interior. The oxygen transport parameter also increased with age of barley, indicating easier diffusion of oxygen within the membrane and its higher fluidity. The data are consistent with age-related changes of the spin label parameters obtained directly by EPR spectroscopy. Similar outcome was also observed when senescence was induced in mature secondary barley leaves by dark incubation. Such leaves showed higher membrane fluidity in comparison with leaves of the same age, grown under light conditions. Changes in the membrane fluidity of barley secondary leaves were compared with changes in the levels of carotenoids (car) and proteins, which are known to modify membrane fluidity. Determination of total car and proteins showed linear decrease in their level with senescence. The results indicate that thylakoid membrane fluidity of barley leaves increases with senescence; the changes are accompanied with a decrease in the content of car and proteins, which could be a contributing factor.

A new theoretical approach to simulation of EPR spectra of spin-labels in protein and membrane systems

A certain model of nitroxyl radial movement is used to interpret EPR spectra of spin-labeled products. In the model proposed a fast reorientation of a spin label occurred due to the order parameter, and a slow motion of the carrier of a spin label, already with partially averaged magnetic parameters, is determined by the rotational correlation time of the macromolecule. By the example of different proteins we demonstrate how to get a fairly good coincidence of simulated EPR spectra with experimental ones within that simple model in solution and using an adsorbent. It is minimalistic in sense of involved motion parameters, and thus attempts to minimize the ambiguity of spectra interpretation. We show that TEMPO radical in lipid phase obeys two-motion model, undergoing fast anisotropic reorientation due to being incorporated into axial phase, and, is additionally affected by slow stochastic process characterized by effective nanosecond-scale correlation time, which can be attributed to domain structure of the membrane.

Simulation of electron paramagnetic resonance spectra of spin-labeled molecules from replica-exchange molecular dynamics

We report a general approach for the simulation of the electron paramagnetic resonance (EPR) spectra of spin labels attached to peptides and proteins directly from replica-exchange molecular dynamics (REMD) trajectories. Conventional MD trajectories are generally inadequate for the prediction of EPR line shapes since the label can become trapped in one or more of a set of rotameric states, thus preventing both conformational sampling and accurate estimates of the exchange rates between different rotamers. The advantage of using REMD is its ability to provide both efficient conformational sampling and kinetic information for spin-label dynamics. Our approach is illustrated with spin-labeled peptide. This approach to REMD-EPR simulation paves the way for the wider application of MD modeling to the simulation and interpretation of EPR spectra of spin-labeled molecules.

Ozone-induced oxidative modification of fibrinogen molecules

Ozone-induced oxidation of fibrinogen has been investigated. The conversion of oxidized fibrinogen to fibrin catalyzed either by thrombin or by a reptilase-like enzyme, ancistron, in both cases is accompanied by production of gels characterized by a higher weight/length ratio of fibrils in comparison with the native fibrin gels. IR spectra of the D and E fragments isolated from unoxidized and oxidized fibrinogen suggest a noticeable transformation of functional groups by oxidation. A decrease in content of N-H groups in the peptide backbone and in the number of C-H bonds in aromatic structures, as well as a decrease in the intensity of the C-H valence vibrations in aliphatic fragments CH2 and CH3 were found. The appearance in the differential spectra of the D fragments of rather intense peaks in the interval of 1200-800 cm(-1) clearly indicates the interaction of ozone with amino acid residues of methionine, tryptophan, histidine, and phenylalanine. Comparison of the differential spectra for the D and E fragments suggests that fibrinogen fragment D is more sensitive to the oxidant action than fragment E. Using EPR spectroscopy, differences are found in the spectra of spin labels bound with degradation products of oxidized and unoxidized fibrinogen, the D and E fragments, caused by structural and dynamical modifications of the protein molecules in the areas of localization of the spin labels. The relationship between the molecular mechanism of oxidation of fibrinogen and its three-dimensional structure is discussed.

The effects of general anesthetics on ESR spectra of spin labels in phosphatidylcholine vesicles containing purified Na,K-ATPase or microsomal protein

We investigated the effects of general anesthetics on liposome containing spin labels, 5-doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA), and purified Na,K-ATPase or membrane protein of microsome using an electron spin resonance (ESR) spectroscopy.The spectra of 16-DSA in liposomes with both proteins showed three sharp signals compared with 5-DSA. The difference in the order parameter S value of 5-DSA and 16-DSA suggested that the nitroxide radical location of 5-DSA and 16-DSA were different in the membrane bilayer. The results were almost the same as those obtained in liposomes without proteins.The addition of sevoflurane, isoflurane, halothane, ether, ethanol and propofol increased the intensity of the signals, but the clinical concentrations of anesthetics did not significantly alter the S and τ values, which are indices of the fluidity of the membrane.These results suggest that anesthetics remain on the surface of the lipid bilayer and do not act on both the inside hydrophobic area and the relatively hydrophilic area near the surface. These results and others also suggest that the existence of Na,K-ATPase and microsomal proteins did not affect the environment around the spin labels in the liposome and the effects of anesthetics on liposome as a model membrane.

The Interaction of the Polyene Antibiotic Amphotericin B with DODAB Aggregates: A Structural Characterization

Amphotericin B (AmB) is widely used in the treatment of systemic fungal infections, despite its toxic effects. Nephrotoxicity, ascribed as the most serious toxic effect, has been related to the state of aggregation of the antibiotic. In search of the increase in AmB antifungal activity associated with low toxicity, several AmB - amphiphilic formulations have been proposed. This work focuses on the structural characterization of a specific AmB formulation: AmB associated with sonicated dioctadecyl dimethylammonium bromide (DODAB) aggregates. Here, it was confirmed that sonicated DODAB dispersion is constituted by DODAB bicelles, and that monomeric AmB is much more soluble in bicelles than in DODAB vesicles. A new optical parameter is proposed for the quantitation of amphiphilic-bound monomeric AmB. With theoretical simulations of the spectra of spin labels incorporated in DODAB bicelles, it was possible to prove that monomeric AmB binds preferentially to lipids located at the edges of DODAB bicelles, rigidifying them, and decreasing the polarity of the region. That special binding of monomeric AmB along the borders of bicelles, where the lipids are highly disorganized, could be used in the formulation of other carriers for the antibiotic, including mixtures of natural lipids which are known to form bicelles. Financial support: USP, FAPESP, CNPq

Encapsulation Influence on EPR Parameters of Spin-Labels: 2,2,6,6-Tetramethyl-4-methoxypiperidine-1-oxyl in Cucurbit[8]uril.

Encapsulation of a nitroxide spin label into a host cavity can prolong the lifetime of the spin label in biological tissues and other environments. Although such paramagnetic supramolecular complexes have been extensively studied experimentally, there is yet little understanding of the role of the encapsulation on the magnetic properties of the spin labels and their performance at the atomistic level. In this work, we approach this problem by modeling encapsulation induced changes of the magnetic properties of spin labels for a prototypical paramagnetic guest-host complex, 2,2,6,6-tetramethyl-4-methoxypiperidine-1-oxyl, enclosed in the hydrophobic cavity of cucurbit[8]uril, using state-of-the-art hybrid quantum mechanics/molecular mechanics methodology. The results allow a decomposition of the encapsulation shift of the electronic g-tensor and the nitrogen isotropic hyperfine coupling constant of nitroxide radical into a set of distinct contributions associated with the host cavity confinement and with changes of the local solvent environment of the spin label upon encapsulation. It is found that the hydrophobic cavity of cucurbit[8]uril only weakly influences the electronic g-tensor of the 2,2,6,6-tetramethyl-4-methoxypiperidine-1-oxyl but induces a significant encapsulation shift of the nitrogen hyperfine coupling constant. The latter is caused by the change of topology of the hydrogen bonding network and the nature of the hydrogen bonds around the spin label induced by the hydrophobic cavity of the inclusion host. This indirect effect is found to be more important than the direct influence of the cavity exerted on the radical. The ramification of this finding for the use of approximate methods for computing electron paramagnetic resonance spectra of spin labels and for designing optimal spin labels based on guest-host templates is discussed.

Aqueous dispersions of DMPG in low salt contain leaky vesicles

Aqueous dispersions of dimyristoyl phosphatidylglycerol (DMPG), at low ionic strength, display uncommon thermal behavior. Models for such behavior need to assign a form to the lipid aggregate. Although most studies accept the presence of lipid vesicles in the lipid gel and fluid phases, this is still controversial. With electron spin resonance (ESR) spectra of spin labels incorporated into DMPG aggregates, quantification of [14C]sucrose entrapped by the aggregates, and viscosity measurements, we demonstrate the existence of leaky vesicles in dispersions of DMPG at low ionic strength, in both gel and fluid phases of the lipid. As a control system, the ubiquitous lipid dimyristoyl phosphatidylcholine (DMPC) was used. For DMPG in the gel phase, spin labeling only indicated the presence of lipid bilayers, strongly suggesting that DMPG molecules are organized as vesicles and not micelles or bilayer fragments (bicelles), as the latter has a non-bilayer structure at the edges. Quantification of [14C]sucrose entrapping by DMPG aggregates revealed the presence of highly leaky vesicles. Due to the short hydrocarbon chains (14C atoms), DMPC vesicles were also found to be partially permeable to sucrose, but not as much as DMPG vesicles. Viscosity measurements, with the calculation of the intrinsic viscosity of the lipid aggregate, showed that DMPG vesicles are rather similar in the gel and fluid phases, and quite different from aggregates observed along the gel–fluid transition. Taken together, our data strongly supports that DMPG forms leaky vesicles at both gel and fluid phases.

Influence of Cholesterol and β-Sitosterol on the Structure of EYPC Bilayers

The influence of cholesterol and β-sitosterol on egg yolk phosphatidylcholine (EYPC) bilayers is compared. Different interactions of these sterols with EYPC bilayers were observed using X-ray diffraction. Cholesterol was miscible with EYPC in the studied concentration range (0-50 mol%), but crystallization of β-sitosterol in EYPC bilayers was observed at X ≥ 41 mol% as detected by X-ray diffraction. Moreover, the repeat distance (d) of the lamellar phase was similar upon addition of the two sterols up to mole fraction 17%, while for X ≥ 17 mol% it became higher in the presence of β-sitosterol compared to cholesterol. SANS data on suspensions of unilamellar vesicles showed that both cholesterol and β-sitosterol similarly increase the EYPC bilayer thickness. Cholesterol in amounts above 33 mol% decreased the interlamellar water layer thickness, probably due to "stiffening" of the bilayer. This effect was not manifested by β-sitosterol, in particular due to the lower solubility of β-sitosterol in EYPC bilayers. Applying the formalism of partial molecular areas, it is shown that the condensing effect of both sterols on the EYPC area at the lipid-water interface is small, if any. The parameters of ESR spectra of spin labels localized in different regions of the EYPC bilayer did not reveal any differences between the effects of cholesterol and β-sitosterol in the range of full miscibility.

Structural characterization of the interaction of the polyene antibiotic Amphotericin B with DODAB bicelles and vesicles

Amphotericin B (AmB) is widely used in the treatment of systemic fungal infections, despite its toxic effects. Nephrotoxicity, ascribed as the most serious toxic effect, has been related to the state of aggregation of the antibiotic. In search of the increase in AmB antifungal activity associated with low toxicity, several AmB-amphiphile formulations have been proposed. This work focuses on the structural characterization of a specific AmB formulation: AmB associated with sonicated dioctadecyl dimethylammonium bromide (DODAB) aggregates. Here, it was confirmed that sonicated DODAB dispersion is constituted by DODAB bicelles, and that monomeric AmB is much more soluble in bicelles than in DODAB vesicles. A new optical parameter is proposed for the estimation of the relative amount of amphiphile-bound monomeric AmB. With theoretical simulations of the spectra of spin labels incorporated in DODAB bicelles it was possible to prove that monomeric AmB binds preferentially to lipids located at the edges of DODAB bicelles, rigidifying them, and decreasing the polarity of the region. That special binding of monomeric AmB along the borders of bicelles, where the lipids are highly disorganized, could be used in the formulation of other carriers for the antibiotic, including mixtures of natural lipids which are known to form bicelles.

End-group distributions of multiple generations of spin-labeled PAMAM dendrimers.

Dendrimers are attractive templates to display functional molecular components. Since the behavior of dendrimer systems can depend greatly on the accessibility of these molecular components to the external environment, and on the spatial arrangement of functional groups attached to the dendrimer terminal branches (end-groups), techniques to determine the locations of end-groups are highly desirable. In this report, we describe a method to analyze the EPR spectra of multiple generations of poly(amidoamine) (PAMAM) dendrimers which have spin-labels attached to end-groups in variable percentages of the total number of available sites. The spectra are treated as a convolution of a narrow spin-label spectrum and a variable line broadening function. Trends in the parameters that describe the best-fit line broadening function with spin-label loading reveal the spatial arrangements and homogeneity of spin environments of the labels. We observe a shift in the end-group distribution from generation 3 (G(3)) to G(4) dendrimers that indicates a change in morphology from an open, extended structure to a more dense, compact arrangement.

Variable Coupling Scheme for High-Frequency Electron Spin Resonance Resonators Using Asymmetric Meshes

The sensitivity of a high frequency electron spin resonance (ESR) spectrometer depends strongly on the structure used to couple the incident millimeter wave to the sample that generates the ESR signal. Subsequent coupling of the ESR signal to the detection arm of the spectrometer is also a crucial consideration for achieving high spectrometer sensitivity. In previous work, we found that a means for continuously varying the coupling was necessary for attaining high sensitivity reliably and reproducibly. We report here on a novel asymmetric mesh structure that achieves continuously variable coupling by rotating the mesh in its own plane about the millimeter wave transmission line optical axis. We quantify the performance of this device with nitroxide spin-label spectra in both a lossy aqueous solution and a low loss solid state system. These two systems have very different coupling requirements and are representative of the range of coupling achievable with this technique. Lossy systems in particular are a demanding test of the achievable sensitivity and allow us to assess the suitability of this approach for applying high frequency ESR to the study of biological systems at physiological conditions, for example. The variable coupling technique reported on here allows us to readily achieve a factor of ca. 7 improvement in signal to noise at 170 GHz and a factor of ca. 5 at 95 GHz over what has previously been reported for lossy samples.

Abstract: S3-29 MULTIPLE MUTATIONS PERTURB A SMALL NUMBER OF PATHWAYS IN AN HDL MUTAGENESIS SCREEN

The ESR spectra of cholestane spin labels (CSL) in dioleoylphosphatidylcholine (DOPC) bilayers containing 20 wt% of cholesterol, 7-dehydrocholesterol, β-sitosterol, stigmasterol and lanosterol exhibit a marked similarity, thus indicating that these steroids induced the same effects on the lipid bilayer over the temperature range 21–55°C. The incorporation of these steroids into the DOPC bilayers enhances the orientational order of the CSL molecules at every temperature studied, but only induces a pronounced slow-down in their rotational motions at temperatures above 35°C. Similar results were obtained in DOPC/ergosterol multilamellar liposomes, but the changes are now less pronounced than in the other five DOPC/steroid systems. In contrast, the addition of stigmasterol to digalactosyldiacylglycerol (DGDG) bilayers appears to increase the order parameter 〈P2〉, without affecting the diffusion coefficients.Furthermore, the incorporation of 7-dehydrocholesterol to DGDG bilayers causes a large enhancement in the orientational order, but has only a small effect on D⊥ of the CSL molecules. Importantly, this latter effect appears to be independent of temperature. The marked changes in the rates of the rotational motion brought about by the addition of steroids, contrasts with the lack of a significant effect of unsaturation on the bilayer dynamics reported by us previously (Korstanje et al. (1989), Biochim. Biophys. Acta 980, 225–233, and 982, 196–204).

Head and Rod 1 Interactions in Vimentin

We have used site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) to identify residues 17 and 137 as sites of interaction between the head domain and rod domain 1A of the intermediate filament protein vimentin. This interaction was maximal when compared with the spin labels placed at up- and downstream positions in both head and rod regions, indicating that residues 17 and 137 were the closest point of interaction in this region. SDSL EPR characterization of residues 120-145, which includes the site of head contact with rod 1A, reveals that this region exhibits the heptad repeat pattern indicative of alpha-helical coiled-coil structure, but that this heptad repeat pattern begins to decay near residue 139, suggesting a transition out of coiled-coil structure. By monitoring the spectra of spin labels placed at the 17 and 137 residues during in vitro assembly, we show that 17-137 interaction occurs early in the assembly process. We also explored the effect of phosphorylation on the 17-137 interaction and found that phosphorylation-induced changes affected the head-head interaction (17-17) in the dimer, without significantly influencing the rod-rod (137-137) and head-rod (17-137) interactions in the dimer. These data provide the first direct evidence for, and location of, head-rod interactions in assembled intermediate filaments, as well as direct evidence of coiled-coil structure in rod 1A. Finally, the data identify changes in the structure in this region following in vitro phosphorylation.

Calculation of the EPR Spectrum of a Small Nitroxide from Molecular Dynamics Simulations

Algorithms have been developed for calculating the continuous wave electron paramagnetic resonance (EPR) spectrum of a nitroxide spin label from a free-induction decay (FID) derived from a molecular dynamics (MD) simulation. These algorithms have been successfully used to simulate the EPR spectra of two spin-labeled mutants of T4 lysozyme, T4L F153R1 and T4L K65R1 [DeSensi et al., Biophys. J. 94, 3798-3809 (2008)]. In this previous work, MD simulations were performed using the AMBER suite of programs, the all-atom AMBER99 force field, the particle mesh Ewald (PME) method for treating long-range electrostatic interactions, and the SPC/E water model. In order to test the effect of the water model used in the AMBER-based molecular dynamics simulations, calculations for a small nitroxide [3-hydroxymethyl-(1-oxy-2,2,5,5-tetramethylpyrroline) or 3MeOHSL] are being performed using a variety of different water models (SPC/E, TIP3P, TIP4P). The EPR spectra calculated from these molecular dynamics simulations of 3MeOHSL will be compared to experimental data.

Dynamics and Local Ordering of Spin-Labeled Prion Protein: An ESR Simulation Study of a Highly PH-Sensitive Site

Valine 160 on β-sheet-2 (S2) of mouse prion (moPrPC) has been previously identified as the most highly pH-sensitive site on moPrPC by ESR spectroscopy using site-directed spin labeling (SDSL) technique. However, no further theoretical analysis to reveal the molecular dynamics reported on the experimental ESR spectra is available. The X-band ESR spectra of R1 nitroxide spin label at V160 and four other sites are carefully analyzed over large pH and temperature ranges using a spectral simulation method based upon stochastic Liouville equation (SLE). The results clearly reveal the dynamics and ordering of the local environment of V160R1 showing that (i) molecular mobility of V160R1 on S2 gradually increases with a decrease of pH from 7.5 to 4.5; (ii) two distinctly different spectral components are simultaneously present in all spectra of V160R1 studied. The spectral components are, respectively, denoted as immobile (Im), characterized by lower molecular mobility and higher ordering, and mobile (Mb) component of high mobility and low ordering. The population ratio (Im/Mb) increases with increasing pH, while Im remains dominant in all V160R1 spectra. It suggests a more mobile and disordered dynamic molecular structure for mouse PrPC, which is very likely correlated with increased β-sheet content at low pH, as the environment changes from neutral to acidic pH. Together with the results of the SLE-based analyses on the spectra of other sites that appear pH-insensitive, we suggest that the simultaneous presence of the spectral components for V160R1 is strongly correlated with the coexistence of multiple protein conformations in local structure of PrPC over the varied pH range. It demonstrates that the combined approach of the SDSL technique and the SLE-based analysis leads to a powerful method for unraveling the complexity of protein dynamics.