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PubMed1982

Transmembrane electrical currents of spin-labeled hydrophobic ions.

When spin-labeled phosphonium ions are rapidly mixed with phospholipid vesicles, time-dependent changes in the electron paramagnetic resonance spectrum of the spin label are observed. These changes are interpreted in terms of transmembrane transport of the hydrophobic ion, and simple analysis of the data at different membrane potentials is shown to give the binding constant of the ion to both membrane surfaces, the permeability, and current-voltage relationship for the vesicle membrane in the presence of the hydrophobic ion. These results establish the time resolution for methods using the phosphonium ion as a probe of time-dependent potentials across vesicle membranes, as well as provide fundamental information regarding the binding and transport of hydrophobic cations across bilayers. This latter point is significant in view of the fact that hydrophobic cations have not been well characterized in planar bilayers due to their weak binding and low conductance.

Lipid Bilayers↗
PubMed1976

Electron spin resonance spin-label studies of intercalation of ethidium bromide and aromatic amine carcinogens in DNA.

A spin-labeled technique has been developed for the study of carcinogen-DNA intercalation mechanisms utilizing a modified ethidium bromide as a model ligand and specifically synthesized spin labels of carcinogenic aromatic amines such as 2-aminofluorene, 2-aminoanthracene, and 6-aminochrysene. The technique takes advantage of the characteristic electron spin resonance anisotropy of the corresponding spin labels complexed with DNA. Analysis of the orientation-dependent information thus obtained has clearly shown that the lignad moieties in all 4 respective complexes are accommodated in adjacent base-pair layers of the DNA with the nitroxide reporter oriented in 1 preferential direction. The geometry of the nitroxide rings with respect to their respective constraining ligands in a particular complex appeared to be largely dependent upon the size of the ligand moiety and the position of the reporter attachment on the ligand molecule. A coplanar configuration is most favored for the spin labels in which the ligand moiety per se is a 3-fused ring with the nitroxide substituted at a position on or close to the longitudinal end of the ligand molecule. Temperature studies in which the heat-induced release of the bound label is monitored by electron spin resonance gives an accurate measure of the complex dissociation characteristics that appear to parallel the helical coil cooperative melting transition monitored by absorbance measurements. The extent of dissociation of the bound spin labels parallels almost exactly the melting, i.e., unzipping of the DNA double helix, suggesting that the spin labels are immobilized by intercalation at the site on the DNA directly involved in the maintenance of the double helical structure. Analysis of the electron spin resonance spectra of the complexes in terms of the extent of immobilization indicates that, for aromatic amine spin labels, two different binding species exist, one strongly immobilized presumably at a guanine-cytosine base-pair region and the other partially immobilized in a region other than guanine-cytosine base pair. Competitive binding of the labels with respect to their parent ligand and binding isotherm results show that nitroxide substitution does not affect the type of binding but does affect the extent of binding, i.e.. binding constant with the labeled compound having a lower affinity.

Amines↗
PubMed1976

Study of the transverse diffusion of spin-labeled phospholipids in biological membranes. II. Inner mitochondrial membrane of rat liver: use of phosphatidylcholine exchange protein.

Spin-labeled phosphatidylcholine was incorporated into the membrane of isolated "inner membrane+matrix" particles of rat liver mitochondria by incubation with sonicated spin-labeled phosphatidylcholine vesicles at 22 degrees C. When the spin label was on the acyl chain the incorporation of phosphatidylcholine into the membrane was stimulated by the presence of the phosphatidylcholine exchange protein extracted from rat or beef liver. On the other hand no stimulation was observed when the nitroxide was on the polar head-group. When spin-labeled phosphatidycholine was incorporated into the mitochondrial membrane in the absence of phosphatidylcholine exchange protein, ascorbate treatment at 0 degrees C reduced the EPR signal of the spin-labeled membranes by approximately 50%, indicating that fusion incorporates molecules equally on both sides of the membrane. On the other hand when spin-labeled phosphatidylcholine was incorporated in the presence of the exchange protein most of the EPR signal could be destroyed by the ascorbate treatment at 0 degrees C, indicating that the spin-labeled phosphatidylcholine had been selectively incorporated in the outer layer of the membrane. Finally when the label is on the polar head-group the inner content of mitochondria reduces the label facing the matrix, thus creating again an anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the mitochondrial membrane was found to be stable at 25 degrees C for more than 2 h. It is therefore concluded that the rate of outside-inside and inside-outside transitions are extremely slow (half-life greater than 24 h).

Animals↗
PubMed1986

Redox-active daunomycin-spin-labeled nucleic acid complexes.

Interaction studies between daunomycin (DM) and enzymatically spin-labeled nucleic acid duplexes reveal two modes of binding by electron spin resonance (ESR) spectroscopy. At a low drug/nucleotide (D/N) ratio, the drug binds in the intercalative mode with only a slight reduction in base mobility. Saturation in the intercalative mode is achieved at a lower D/N ratio for B' DNA than for B DNA. After full intercalation, further addition of DM seems to destabilize the helix and to allow the formation of redox-active DM stacks complexed to the nucleic acid lattice. These stacks will irreversibly oxidize all the nitroxides covalently bound to the 4- or 5-position of the pyrimidine base. Interactions between DM and spin-labeled single-stranded nucleic acids lead directly to the formation of redox-active complexes, while mixing of the drug with spin-labeled nucleic acid building blocks not incorporated in a nucleic acid lattice causes no ESR signal change. Complete disappearance of the ESR signal of spin-labeled nucleic acids extrapolates to a D/N value which is a constant for a particular lattice system and is independent of spin-labeling content.

DNA↗
PubMed1977

Spin-label studies on the aqueous regions of phospholipid multilayers.

Water-soluble spin labels were used to study dimyristoyllecithin (DML) phospholipid multilayers. Previous studies report that there is a "bound" water region associated with dimyristoyllecithin containing about 10 molecules of water per phospholipid, a "trapped" water region located between the lamellae containing approximately 11 molecules per phospholipid, and a "ftion show that certain water-soluble spin-label mol-cules have their motional properties differentially modified by these three water environements. Furthermore, the labels also reveal the onset of lipid-phase transitions even though they have high water solubility. A phosphate-containing spin label demonstrated strong an isotropic motion in the lipid-water system above the phase transition but not below. The addition of cholesterol to the DML-water system removed the anisotropic motion of 2,2,6,6-tetramehtyl-4-phosphopiperidine-N-oxyl (Tempophosphate) and obscured the detection bound, trapped, and free water. In addition to the change-charge interactions between Tempophosphate and DML, two other spin labels were used both in the charged and uncharged states. 2,2,6,6-Tetramethyl-4-aminopiperidine-N-oxyl (Tempamine) in the charged state showed extremely strong anisotropic motion, presumably due to the interaction between the charged amine and the phosphate group of DML. When only partially charged, Tempamine showed much less anisotropic motion. PCA was analyzed at pH values where the carboxyl group was protonated and unprotonated. The resulting interaction was different at the two pH values. These water-soluble spin labels mimic ionic or nonionic solutes. Upon freezing, the spin labels are shown to be expelled from the ice regions into the remaining aqueous regions. The usefulness of this approach in studying solute behavior when freezing occurs and potential studies involving aqueous regions of cytoplasm are considered.

Binding Sites↗
PubMed2006

Time-resolved electron spin resonance studies of spin-labelled lipids in membranes.

Recently, developments in time-resolved spin-label electron spin resonance (ESR) spectroscopy have contributed considerably to the study of biomembranes. Two different applications of electron spin echo spectroscopy of spin-labelled phospholipids are reviewed here: (1) the use of partially relaxed echo-detected ESR spectra to study the librational lipid-chain motions in the low-temperature phases of phospholipid bilayers; (2) the use of electron spin echo envelope modulation spectroscopy to determine the penetration of water into phospholipid membranes. Results are described for phosphatidylcholine bilayer membranes, with and without equimolar cholesterol, that are obtained with phosphatidylcholine spin probes site-specifically labelled throughout the sn-2 chain.

Cholesterol↗
PubMed2003

Molecular mechanisms of gene regulation studied by site-directed spin labeling.

The technique of site-directed spin labeling using cysteine substitution mutagenesis followed by modification of the sulfhydryl group with a nitroxide reagent is emerging as a valuable alternative for the determination of protein folds and conformational changes in a variety of systems. The incorporation of pairs of nitroxides allows determination of intramolecular distances and distance changes with a spatial resolution at the level of the backbone fold under conditions relevant to function. The methodology of electron paramagnetic resonance spectral data acquisition and interpretation is reviewed with studies on conformational changes of Tet repressor (TetR) and the human immunodeficiency virus type 1 reverse transcriptase (RT) on interaction with nucleic acid substrates or inhibitors in solution. A twisting motion of the DNA reading heads of TetR on induction by tetracycline (tc) is observed in solution by changes of the interspin distances between interacting nitroxides at positions 22/22(') or 47/47('). Spin-label side chains located near the tc-binding pocket or at position 202 indicate different conformations for the tc- and DNA-complexed repressor also in the core of the protein. Interspin distances between spin-labeled residue positions 24 and 287 in the fingers and the thumb domains of RT complexed with dsDNA or a pseudoknot RNA in solution were found to agree with the respective crystal data of the so-called open and closed conformations. For the unliganded RT a temperature-dependent equilibrium between these two states is observed.

Electron Spin Resonance Spectroscopy↗
PubMed1991

Spatial arrangement of coenzyme and substrates bound to L-3-hydroxyacyl-CoA dehydrogenase as studied by spin-labeled analogues of NAD+ and CoA.

The synthesis of nitroxide spin-labeled derivatives of S-acetoacetyl-CoA, S-acetoacetylpantetheine, and S-acetoacetylcysteamine is described. These compounds are active substrates of L-3-hydroxyacyl-CoA dehydrogenase [(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.35] exhibiting vmax values from 20% to 70% of S-acetoacetyl-CoA itself. S-Acetoacetylpantetheine and S-acetoacetylcysteamine form binary complexes with the enzyme and exhibit ESR spectra typical for immobilized nitroxides. In the case of spin-labeled pantetheine, the radical is more mobile. When spin-labeled substrates are bound simultaneously to each active site of this dimeric enzyme, spin-spin interactions differentiate between two alternate orientations of the substrate [Birktoft, J.J., Holden, H.M., Hamlin, R., Xuong, N.H., & Banaszak, L.J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8262-8266]. The fatty acid moiety is thought to be located in a cleft between two domains whereas a large part of the CoA moiety probably extends into the solution. NAD+, spin-labeled at N6 of the adenine ring, is an active coenzyme of L-3-hydroxyacyl-CoA dehydrogenase (60% vmax). Complexes with the enzyme exhibit ESR spectra typical of highly immobilized nitroxides. Binding of coenzyme NAD+ causes conformational changes of the binary enzyme/substrate complex as revealed by changes in the ESR spectrum of spin-labeled S-acetoacetylpantetheine.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1977

Surface localization of sites of reduction of nitroxide spin-labeled molecules in mitochondria.

The relative rates of reduction of several spin-labeled molecules that partition differently across the hy-drophobic-interface of inner membranes from rat liver mitochondria were investigated. Spin labels localized either deep in the hydrophobic region or in the aqueous phase are only slowly reduced; however a spin-labeled analogue of the cationic detergent cetyltrimethylammonium bromide that partitions at the interface is rapidly reduced by coupled electron transport. Chemical studies on the reduction and oxidation of the spin label show that loss of signal is due to reduction and not destruction of the label. No evidence was found for flip-flop of the label in submitochondrial preparations. Spin reduction of respiring mitochondria, mitoplasts, or inverted submitochondrial preparations is inhibited by rotenone but is relatively insensitive to antimycin A and KCN. Because the midpoint potentials of the spin labels were found to be similar to that of ubiquinone, it is concluded that reducing equivalents of mitochondrial electron transport from this region of the chain are channeled to either membrane interface.

Aerobiosis↗
PubMed1982

Interaction of nitroxide spin labels with chloroplasts.

Chloroplasts isolated from oats eliminated the electron spin resonance (ESR) signals from spin labels in white light and partially restored them in far-red light. Only the white light-mediated reaction was blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In contrast, oat (Avena sativa L. cv. Garry and Park) leaf mesophyll protoplasts oxidized the spin labels in both white and far-red light, with and without DCMU. Light had no obvious effect on spin label motion within chloroplast membranes. The results suggest that, in isolated chloroplasts, nitroxide spin labels may be reduced by photosystem I within the thylakoid bilayer resulting in loss of the ESR signals. The reduced forms may be reoxidized by an element of the photosynthetic electron transport chain which operates between the DCMU block and the photosystem I reaction center. In addition, a light-mediated destruction of the spin labels occurs in both chloroplasts and protoplasts. The reduced form of the nitroxide (i.e. the hydroxylamine) may be resistant to this destruction.

Journal Article↗
PubMed1998

Determination of high-spin iron(III)-nitroxyl distances in spin-labeled porphyrins by time-domain EPR.

Continuous wave EPR spectra of the nitroxyl signals for four spin-labeled high-spin (h.s.) Fe(III) porphyrins showed partially resolved splittings at temperatures near 4 K. Axial ligands were fluoride, chloride, or bromide. As temperature was increased to 20 to 30 K the iron-nitroxyl splitting collapsed due to increasing rates of iron relaxation. Electron spin-echo (ESE) spectroscopy showed that above about 6 K collapse of the iron-nitroxyl spin-spin splitting caused a dramatic increase in the nitroxyl phase memory relaxation rates. Electron spin relaxation rates were determined for Fe(tetratolylporphyrin)X, X = F, Cl, Br, in toluene solution by ESE or inversion recovery at 4.5 to 6 K and by analysis of the temperature-dependent contributions to the continuous wave EPR linewidths between about 10 and 120 K. Above about 10 K iron relaxation rates increase in the order X = F < Cl < Br, which is the order of increasing zero-field splitting. Saturation recovery data for two spin-labeled h.s. iron(III) porphyrins between about 15 and 120 K and for two additional spin-labeled h.s. iron(III) porphyrins between about 85 and 120 K demonstrated that interaction with the h. s. iron enhanced the electron spin relaxation rate of the spin label. The saturation recovery curves for the nitroxyl were analyzed to determine interspin distances using a modified version of the Bloembergen equation and independently determined iron relaxation rates. Interspin distances were between 11.6 and 15.0 A, were independent of axial ligand, and were in good agreement with values obtained previously for low-spin Fe(III) and Cu(II) analogs.

Algorithms↗
PubMed1987

Adsorbed behavior of spin-labeled silane coupling agent on colloidal silica studied by electron spin resonance.

The adsorption mechanism of silane coupling agent on the colloidal silica surface is studied with spin-labeled silane coupling agent using ESR method. The density of the adsorbed silane molecules was determined to be approximately 1.5-2 molecules per 100 A2 of the silica surface in 95% ethanol solution. In addition, ESR signal of spin-labeled agent adsorbed on the colloidal silica surface was composed of two components; slow component attributable to the agents adsorbed directly on the Si--OH groups of the silica surface through hydrogen bond formation and rapid one attributable to the agents interacted with such adsorbed silane molecules.

Acrylic Resins↗
PubMed1976

Synthesis of biologically active spin-labelled radioactive cytidine diphosphodiglyceride, a novel probe for biological membranes.

A versatile synthesis of spin-labelled radioactive cytidine diphospho-sn-1,2-diacylglycerol (CDP-diglyceride) has been developed based on the combination of the enzymatic acylation of radioactive sn-glycero-3-phosphate with 12-doxyl stearic acid and the chemical conversion of the thus obtained spin-labelled radioactive phosphatidic acid with cytidine monophosphomorpholi-date into spin-labelled radioactive CDP-diglyceride. The method for the isolation and purification of the latter compound was described. This obtained CDP-[2-3H]diglyceride contained 10% of fatty acids of paramagnetic nature, presumably present as a covalently bound 12-doxyl stearic acid esters. The biological activity was tested by using the synthesized compound as a substrate in the mitochondrial biosynthesis of phosphatidylglycerol. It was found that spin-labelled CDP-[2-3H]diglyceride prepared as described can be converted in the presence of sn-[2-14C]-glycero-3-phosphate into a spin-labelled [2-3H, 2'-14C]phosphatidylglycerol with isolated rat liver mitochondria, establishing therefore that the site of its utilization is identical with the site of phosphatidylglycerol synthesis in isolated mitochondria, i.e. inner mitochondrial membrane. Results described demonstrate that the synthesized spin-labelled CDP-diglyceride can be used as a specific probe for the spin- and radioactive covalent labelling of polyglycerophosphatides of mitochondrial membranes. Some implications and further possibilities in the study of biological membranes using the spin-labelled radioactive CDP-diglyceride are discussed.

Animals↗
PubMed1990

Subcellular distribution of a nitroxide spin-labeled netropsin-acridine hybrid in living KB cells: Electron Spin Resonance Study.

NETGA is an hybrid derivative which possesses an intercalating heterocyclic nucleus related to amsacrine and a minor groove binding squeletton related to netropsin. Cellular uptake of this drug has been studied by Electron Spin Resonance (ESR) spectroscopy using a spin-label derivative of NETGA (SL-NETGA). ESR determination of the kinetics of the drug repartition between the cytoplasm and nucleus showed that NETGA accumulated very rapidly and predominantly in the nucleus. Analysis of the anisotropic ESR spectra recorded in the nuclear compartment are in agreement with a strong binding of the drug to the DNA besides confirmed by a maximum delta Tm of 12 degrees C between the spin-label compound-DNA complex and the DNA alone.

Acridines↗
PubMed2001

Evaluation of site-directed spin labeling for characterizing protein-ligand complexes using simulated restraints.

Simulation studies have been performed to evaluate the utility of site-directed spin labeling for determining the structures of protein-ligand complexes, given a known protein structure. Two protein-ligand complexes were used as model systems for these studies: a 1.9-A-resolution x-ray structure of a dihydrofolate reductase mutant complexed with methotrexate, and a 1.5-A-resolution x-ray structure of the V-Src tyrosine kinase SH2 domain complexed with a five-residue phosphopeptide. Nitroxide spin labels were modeled at five dihydrofolate reductase residue positions and at four SH2 domain residue positions. For both systems, after energy minimization, conformational ensembles of the spin-labeled residues were generated by simulated annealing while holding the remainder of the protein-ligand complex fixed. Effective distances, simulating those that could be obtained from (1)H-NMR relaxation measurements, were calculated between ligand protons and the spin labels. These were converted to restraints with several different levels of precision. Restrained simulated annealing calculations were then performed with the aim of reproducing target ligand-binding modes. The effects of incorporating a few supplementary short-range (< or =5.0 A) distance restraints were also examined. For the dihydrofolate reductase-methotrexate complex, the ligand-binding mode was reproduced reasonably well using relatively tight spin-label restraints, but methotrexate was poorly localized using loose spin-label restraints. Short-range and spin-label restraints proved to be complementary. For the SH2 domain-phosphopeptide complex without the short-range restraints, the peptide did not localize to the correct depth in the binding groove; nevertheless, the orientation and internal conformation of the peptide was reproduced moderately well. Use of the spin-label restraints in conjunction with the short-range restraints resulted in relatively well defined structural ensembles. These results indicate that restraints derived from site-directed spin labeling can contribute significantly to defining the orientations and conformations of bound ligands. Accurate ligand localization appears to require either a few supplementary short-range distance restraints, or relatively tight spin-label restraints, with at least one spin label positioned so that some of the restraints draw the ligand into the binding pocket in the latter case.

Binding Sites↗
PubMed1984

Spin-label studies of erythrocyte deformability. IV. Relation of electron spin resonance spectral change with deformation and orientation of erythrocytes in shear flow.

Electron spin resonance (ESR) spectra of spin-labeled human erythrocytes in shear flow are simulated to derive semi-empirical relations of the ESR spectral change with deformation and orientation of the cells by using a modified theoretical model developed for deformation and orientation of liquid drops. The six observed spectra at different shear stress values were simultaneously simulated by adjusting only two parameters. One parameter can be related to the ratio of the internal to the external viscosity, and the other to the elastic property of the cell membrane. From these results we have derived a semi-empirical relationship between the average deformation index or the orientation angle with a spectral measure, which characterizes the spectral shape change induced by shear stress. Thus, it becomes possible to obtain improved quantitative information on the rheological behavior of red blood cells by using the spin-label ESR method.

Electron Spin Resonance Spectroscopy↗
PubMed2001

Hydrophobic interactions between spin-label 5-SASL and humic acid as revealed by ESR spectroscopy.

The spin-label probe 5-SASL (stearic acid spin-label with nitroxide free radical in position 5 of hydrocarbon chain), detectable by electron spin resonance (ESR), was tested to evaluate pH and reaction time dependencies of hydrophobic interactions with humic acid (HA). Strong changes were observed in 5-SASL ESR spectra in the presence of HA suspensions below pH 5, with disappearance of the three isotropic narrow hyperfine lines of the nitroxide group (typical of free spin-label) and formation of "immobilized" 5-SASL spectra. These changes were interpreted as due to 5-SASL bonding with hydrophobic groups of HA, by van der Waals forces and/or hydrogen bonds, in very hydrophobic sites (probably water-protected) existent in HA below pH 5. However, such sites are absent above pH 5, as demonstrated by a specific experiment to check 5-SASL spectra reversibility. On the other hand, the HA suspension was more efficient in dissolving 5-SASL than water above pH 5. This fact also suggests the existence of "surface" hydrophobic sites, where the spin-label binds to HA while maintaining the nitroxide group in contact with water, as evidenced by the typical free spin-label spectrum and hyperfine interaction splitting (a0 = 1.574 mT). Also experiments checking 5-SASL reversibility bonding with HA were consistent with the supramolecular association model to HA.

Electron Spin Resonance Spectroscopy↗
PubMed1983

Spin label study of erythrocyte deformability. III. Further characterizations of electron spin resonance spectral change in shear flow.

It is demonstrated that the change in the spin label ESR spectrum induced by shear flow reflects the whole cell deformation as a function of the cell surface area-to-volume ratio (s/v), the morphology and intracellular viscosity. Since the effect of the change in the membrane mechanical property on the ESR spectrum has been described previously, the spin label ESR spectrum is now shown to contain full information concerning the whole cell deformability which is determined by the major intrinsic and extrinsic properties of the red blood cells. The result of microphotographic observations shows also that the cells in the flow are elongated and aligned approximately along the flow direction to an increasing extent as the cells flow near the surface of the flat channel walls. Thus, the entire observation confirms the view that the ESR spectral difference-shear rate profile is closely related to the elongation ratio shear rate characteristics obtained by other (optical) methods.

Blood Viscosity↗