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PubMed1996

Spin-labeled nucleotide mobility in the boundary of the EcoRI endonuclease binding site.

A complex consisting of the EcoRI endonuclease site-specifically bound to spin-labeled DNA 26mers was prepared to provide a model system for studying possible conformational changes resulting from protein binding. EPR was used to monitor the mobility of the spin labels that were strategically placed in position 6, 9, or 11 with respect to the dyad axis of the 26mer. These positions are located within the flanking region on either side of the EcoRI hexamer binding site. This allows the monitoring of potential distal structural changes in the DNA helix caused by protein binding. The spectral line shapes indicate that the spin label closest to the EcoRI endonuclease binding site, i.e., in position 6, is most influenced by the binding event. The EPR data are analyzed according to a model that distinguishes between spectral effects due to a change in the hydrodynamic shape of the complex and those resulting from local variations in the spin-label mobility as characterized by a local order parameter S. S reflecting the motional restriction of the spin-labeled base is 0.20 +/- 0.01 for all three oligomers as well as for the two complexes with the label in position 9 or 11, while the position 6 labeled complex yields S = 0.25. To further evaluate the origin of the slightly larger EPR effect observed with position 6 labeled material, molecular dynamics (MD) simulations were used to explore the space accessible to the probes in positions 6, 9, and 11. MD results gave similar nitroxide trajectories for all three labeled 26mers in the absence or presence of EcoRI. Thus, the small position 6 effect is attributed to a structural distortion in the major groove of the DNA at this location possibly corresponding to a bend induced by protein binding. The observation that the spectral changes are small indicates the absence of any significant structural disruption being propagated along the helix as a result of protein binding. Also, the fact that the line shape of the 26mers did not change as expected from hydrodynamic theory in view of the significant increase in molecular volume upon protein binding suggests that there are additional relaxation processes involving the protein and nucleic acid.

Binding Sites↗
PubMed1983

Effect of chlorpromazine on proteins in human erythrocyte membranes as inferred from spin labeling and biochemical analyses.

ESR spectra of erythrocyte membranes labeled with a maleimide spin label (MSL) show two types of label environment: a weakly immobilized component and a strongly immobilized component. Chlorpromazine (CPZ) markedly altered the spectra: at pH 8.0, 3 mM CPZ reduced the amplitude of the spectrum by 40%, and the weakly immobilized component was almost completely removed. In order to clarify the mechanisms of these spectral changes the protein release from erythrocyte membranes induced by CPZ has been followed. CPZ had a weak solubilizing effect on erythrocyte membranes: less than 1% of the membrane protein was released, mainly Band 6. By comparison with the protein release induced by low-salt treatment it was found that the "detergent-like" property of CPZ cannot explain the alterations in the ESR spectra. The nature of the spectral changes induced by CPZ was different from that of changes induced by lowering the pH to 4.5; correlated with other data this shows that changes in organization or conformation of membrane protein cannot explain the CPZ-induced alterations in the ESR spectra. These spectral changes appeared to be due to the reduction by CPZ of the nitroxide free radical. This was documented by the marked reduction of spin concentration of the labeled ghosts in the presence of CPZ resulting in a decrease in amplitude of the ESR spectrum of MSL-labeled erythrocyte ghosts induced by CPZ. The reduction by CPZ of the nitroxide free radical was compared with that induced by ascorbate. It was found that CPZ preferentially reduces the mobile component of the ESR spectrum of MSL-labeled ghosts. The action of CPZ in reducing free radicals may have consequences for patients receiving long-term treatment with phenothiazine derivatives.

Blood Proteins↗
PubMed1976

On the use of the spin labeling technique in the study of erythrocyte membranes.

ESR spectra and scanning electron micrographs of human erythrocytes spin labeled with the conventional stearic acid nitroxide substituted at the 5-position have been obtained over a range of label-to-lipid ratios. While morphological changes as previously reported (Bieri, V. G., Wallach, D. F. H. and Lin, P. S. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4797-4801) are reproduced, it is shown that at label-to-lipid ratios of 1:10 or less the basic ESR spectrum is not significantly affected. At low label concentrations the spin labeling technique is a viable one and can be used to investigate membrane properties.

Binding Sites↗
PubMed2004

Inter- and intra-molecular distances determined by EPR spectroscopy and site-directed spin labeling reveal protein-protein and protein-oligonucleotide interaction.

Recent developments including pulse and multi-frequency techniques make the combination of site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy an attractive approach for the study of protein-protein or protein-oligonucleotide interaction. Analysis of the spin label side chain mobility, its solvent accessibility, the polarity of the spin label micro-environment and distances between spin label side chains allow the modeling of protein domains or protein-protein interaction sites and their conformational changes with a spatial resolution at the level of the backbone fold. Structural changes can be detected with millisecond time resolution. Inter- and intra-molecular distances are accessible in the range from approximately 0.5 to 8 nm by the combination of continuous wave and pulse EPR methods. Recent applications include the study of transmembrane substrate transport, membrane channel gating, gene regulation and signal transfer.

Animals↗
PubMed1993

Transmembrane distribution and translocation of spin-labeled plasmalogens in human red blood cells.

We have synthesized two new spin-labeled alkenylacyl phospholipids (plasmalogens) in order to investigate the transmembrane distribution and transport of this subclass of glycerophospholipids in human red blood cells. The plasmenylethanolamine analogue diffuses rapidly from the outer to the inner leaflet with a half time at 37 degrees C of 30 min comparable to that of the corresponding diacyl-phosphatidylethanolamine spin-label in an ATP-requiring and N-ethyl maleimide sensitive manner. The plateau corresponds to 79% of the aminophospholipids on the inner leaflet. By contrast, after 4 h incubation less than 20% of the plasmenylcholine spin-labels reach the interior. Thus plasmalogens behave as the corresponding diacyl-lipids. We infer that plasmenylethanolamine is transported from the outer to the inner leaflet of the red cell membrane by the aminophospholipid translocase.

Biological Transport↗
PubMed1993

Maleimide, iodoacetamide, indanedione, and chloromercuric spin label reagents with derivatized nitroxide rings as ESR reporter groups for protein conformation and dynamics.

The syntheses of eight nitroxide spin labels which bear maleimide, iodoacetamide, indanedione, or chloromercuric reactive groups and, in addition, a second substituent in the nitroxide ring are presented. The second substituent groups range from hydrophobic and hydrophilic esters to carboxylic acid and secondary and tertiary amine groups. The resulting spin labels are characterized with respect both to protein covalent modification and to the electron spin resonance spectral properties of the bound labels. The effect of the various substituents in the spin label on the reactivity toward the membrane-bound shark rectal gland and pig kidney Na,K-ATPase is described. The spectral differences between immobilized and mobile groups observed by electron spin resonance for the different protein-bound spin labels show that, by selecting an appropriate derivative for modification, a large range of different motional sensitivities of the reporter group can be obtained. Such different series of spin labels should therefore be useful for detecting mobility changes arising from conformational transitions in proteins by conventional electron spin resonance spectroscopy or for measurement of protein rotational diffusion using saturation transfer electron spin resonance spectroscopy. The chloromercuric series is found to be particularly useful because of the high reactivity, the lack of reversibility that potentially is associated with the Michael addition reaction, and the wide range of rotational mobility that is exhibited by the different derivatives.

Animals↗
PubMed1993

Do the spectra of maleimide spin-labelled whole blood platelets reflect the structure and conformation of membrane proteins?

The maleimide spin label (4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl, MSL), the commonly used reagent specific for cysteine thiol groups in proteins, penetrates cell membranes and binds to both the membrane and cytoplasmic protein moieties. In order to differentiate the labelling of these two subpopulations of cell proteins, we developed three different methodological approaches varying in the consequent distribution of this label in platelets. (A) The labelling of platelet proteins was negligible when bovine serum albumin was used in the Tyrode's buffer for the isolation of platelets, as the majority of the spin label was bound to the albumin coated on the platelets. (B) Preblocking of the reactive thiol groups in albumin with non-spin maleimide analog, N-ethylmaleimide (NEM), caused a considerable amount of MSL to bind with whole platelets but the impartment of membrane component was below 50%. It suggests that the majority of the spin label penetrated platelets and was bound to the intrinsic platelet proteins. (C) In order to prevent labelling of intrinsic platelet proteins with MSL, platelets were preincubated with N-ethylmaleimide, which was able to penetrate platelets and block the reactive thiol groups inside the cells. Such a treatment resulted in a saturation of the intrinsic protein residues with this non-spin analog. The subsequent incubation of thus-treated albumin-free platelets with MSL was to enhance considerably the likelihood of the attachment of MSL molecules to the thiol groups available in platelet-membrane proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Heparin modulates conformational states of plasma fibronectin: an electron spin resonance spin label approach.

We have examined the interaction between heparin and human plasma fibronectin using electron spin resonance (ESR) spin label methods. The titratable sulfhydryl groups of plasma fibronectin were modified with a maleimide spin label [Lai and Tooney (1984) Arch. Biochem. Biophys. 228, 465-473]. Addition of heparin resulted in a decrease in the maximum splitting value of the ESR spectrum of spin-labeled fibronectin from 66.8 to 64.3 G, suggesting that heparin induces a conformational alteration of plasma fibronectin. This heparin effect was noticeable at a heparin-to-fibronectin ratio of 20 to 1 and reached a plateau at about 100 to 1. Other sulfated carbohydrates were tested; dextran sulfate was found to be as effective as heparin but chondroitin sulfates were ineffective. The results presented suggest that the binding of heparin changes the molecular conformation of plasma fibronectin to a more relaxed or flexible state.

Electron Spin Resonance Spectroscopy↗
PubMed1996

Interaction of alkanols and local anesthetics with spin-labeled Ca(2+)-ATPase of sarcoplasmic reticulum vesicles.

Alkanols and tertiary amine derivative local anesthetics modify the activity of Ca(2+)-ATPase. In order to investigate the primary binding sites, associated to the functional changes, sarcoplasmic reticulum (SR) Ca(2+)-ATPase was labeled with maleimide derivative spin labels which bind covalently to SH groups of cysteine residues and allow to probe the regions of the protein close to those residues. The EPR measurements showed motional constraints induced by drug-treatment which indicate changes in the enzyme dynamics and structure. n-Alkanols are shown to affect some of the protein-bound labels by restricting their motion. There is, however, no correlation between the functional effects and the observed motional restriction, in the sense that concentrations of the different alcohols leading to the same functional effects do not induce the same degree of restriction. Dibucaine and tetracaine at functional relevant concentrations also restrict the movement of protein bound labels. But, in this case, correlation between spectral changes and functional effects is observed.

1-Butanol↗
PubMed1976

V irus and lectin agglutination of erythrocytes: spin label study of membrane lipid-protein interactions.

Techniques of spin-label electron spin resonance have been used to prove changes in the structure of the lipid phase of erythrocyte membrane after agglutination by viruses and lectins. When chicken erythrocytes are agglutinated by Sendai and influenza viruses and by the lectins concanavalin A and wheat germ agglutinin, the membrane lipid phase becomes more fluid, as detected by three different lipophilic spin-laveled probes. Colchicine, vinblastine, and tetracaine inhibit the fluidization of chicken erythrocyte membrane by Sendai virus, whereas cytochalasin B has no effect. The effect of colchicine was time dependent, the initial inhibition decreasing with longer preincubation times. Extensive treatment of erythrocytes with proteases or neuraminidase, while not altering the bilayer structure, abolishes the effect of Sendai virus on the erythrocyte membrane, suggesting that a change in the interaction of the receptor protein with the lipid phase occurs upon virus attachment. Glutaraldehyde fixation increased the structural rigidity of the chicken erythrocyte membrane and inhibited the effect of viral agglutination. No change in bilayer structure was observed upon agglutination of human erythrocytes or the isolated plasma membranes of either human or chicken erythrocytes. This result is consistent with the drug sensitivity of the effects of agglutination upon chicken erythrocytes, since human erythrocytes and isolated membranes lack microtubule-like structures.

Animals↗
PubMed1992

Spectroscopic and biological studies of spin-labeled tetracycline.

A new nitroxyl labeled tetracycline is synthesized. Proton NMR experiments of tetracycline, spin-labeled tetracycline, and the diamagnetic reduced form in DMSO-d6 are reported. The signals observed in the NMR spectra are all assigned. The NMR data revealed that the spin label is attached to the C-2 amide group on ring A of tetracycline. The spin-labeled tetracycline is also tested in vitro for antitumor activity and is found to be active against leukemia P338/ADR cell line and in melanoma LOX cell line.

Animals↗
PubMed1988

Globoside with spin-labelled fatty acid: bilayer lateral distribution and immune recognition.

We have critically addressed the question of lateral distribution of glycolipids in bilayer membranes, and the effect of glycolipid fatty acid chain length upon such distribution. For this purpose we synthesised the complex neutral glycosphingolipid, globoside, with spin-labelled fatty acid. Base hydrolysis to remove the natural fatty acid was found to deacetylate the GalNAc residue concomitantly, necessitating application of the synthetic route described for gangliosides by Neuenhofer et al. (Biochemistry 24, 525-532 (1985)). Globosides were produced with 18-carbon and 24-carbon fatty acids bearing a spin label at the C-16 position. Spin-labelled globosides were incorporated at 2 and 10 mol% into rigid, highly cooperative bilayer matrices of 1,2-dipalmitoylglycerophosphocholine (DPPC) and also into semi-fluid, non-cooperative membranes of DPPC/cholesterol. Recorded electron paramagnetic resonance (EPR) spectra were analysed by comparison with a library of standards representing samples of known composition. Spectra were manipulated using a computer program which permitted linear combination of standards to stimulate coexistence of laterally separated domains of different composition. The most important conclusions were as follows: (1) at least 80% of the globoside was definitely not confined to domains highly enriched in glycolipid, although there was evidence of binary-phase separation in the rigid DPPC/globoside matrix; (2) the presence of 33 mol% cholesterol reduced the evidence of globoside phase separation; (3) there was remarkably little difference in results whether the globoside fatty acid chain length was similar to that of the phospholipid host matrix or eight carbons longer. Temperature profiles derived over the phase-transition region of DPPC using spin-labelled globoside or an unattached amphiphilic spin label were consistent with these findings. The same systems lent themselves to consideration of the role of glycolipid fatty acid chan length and cholesterol in determining glycolipid crypticity in membranes: (1) polyclonal anti-globoside IgG bound to globoside in DPPC liposomes without inducing agglutination. (2) The same antibodies did agglutinate DPPC/cholesterol liposomes bearing globoside. (3) The effect of cholesterol probably was upon glycolipid dynamics or attitude in the membrane, rather than upon distribution. (4) These observations were basically unaffected by the choice of 18-carbon vs. 24-carbon glycolipid fatty acids.(ABSTRACT TRUNCATED AT 400 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

Evidence for the exchange of arterial spin-labeled water with tissue water in rat brain from diffusion-sensitized measurements of perfusion.

The extraction fraction of vascular water in rat brain is investigated by means of diffusion measurements of arterial spin labeled water at varying cerebral blood flow (CBF) values. The apparent diffusion coefficient (ADC) of the difference of the proton magnetization signal in the brain acquired with and without continuous arterial spin labeling is modeled to provide a measure of the amount of arterial water in tissue and vasculature and thus of the extraction fraction. The tissue and vascular portion of the arterial spin labeled water are differentiated based on their diffusion characteristics in a manner analogous to the intravoxel incoherent motion (IVIM) method. The amount of labeled arterial water that exchanges with tissue water is determined by estimating the fraction of the total signal that is associated with the slow-decaying component of a biexponential fit to the normalized difference signal between the magnetization of brain tissue acquired with and without arterial spin labeling. The results indicate that, at normal CBF (1.15 +/- 0.21 ml x g(-1) x min[-1]), about 90% of the arterial spin labeled water diffuses with an ADC of (1.21 +/- 0.37) x 10[-3] mm2 s[-1]), which is equal to tissue. At high CBF, an increasing fraction of the labeling water has a fast-pseudo-diffusion coefficient due to a decrease in water extraction fractions. The results also show that the contribution of vascular water to the measurement of perfusion by techniques that use endogenous water as a tracer can be efficiently eliminated by the use of diffusion sensitizing gradients with small effective b values (b approximately 20 s/mm2), enabling these techniques to monitor true changes in tissue perfusion.

Animals↗
PubMed1987

Spin-labeled amphotericin B: synthesis, characterization, biological and spectroscopic properties.

A biologically active spin-labeled derivative of amphotericin B has been synthesized by the nucleophilic addition of amphotericin B to 4-(2-iodoacetamido)-2,2',6,6'-tetramethylpiperadine-N-oxyl in dimethyl-sulphoxide at 40 degrees C. The derivative is a moderately water-soluble compound which displays the same biological activity of the parental compound against the sensitive organism Leishmania mexicana; also, the rates of proton-cation exchange induced by the two compounds in large unilamellar liposomes are indistinguishable. The ESR spectra of spin-labeled amphotericin B in lipid vesicles indicate a high degree of motion, very similar to that encountered for the compound in aqueous solutions at neutral pH and in deoxycholate micelles, and suggest that the structures formed by the antibiotic in membranes are composed by a small number of molecules. In contrast, the spectra of the labeled antibiotic in ethanol, diethyl ether and dimethylformamide indicate restricted motion and exchange interactions, probably resulting from the micellar aggregation induced in these media. Ascorbate at 10 mM is able to reduce completely the nitroxide group of the labeled antibiotic in lipid vesicles in less than 30 s, indicating that an asymmetric disposition of the antibiotic molecules across the membrane is capable of inducing its biological and ionophoric properties. Ni2+ and Cu2+ produce moderate exchange broadening of the ESR signal of spin-labeled amphotericin B in lipid vesicles; the comparison of this phenomenom with the exchange broadening produced by the same ions in the ESR spectrum of 2,2',6,6'-tetramethylpiperidine-N-oxyl in water solution suggests an specific Cu2+-amphotericin B interaction in membranes.

Amphotericin B↗
PubMed1982

Localization of spin labels in oat leaf protoplasts.

An assay based on light-mediated oxidation was used to determine whether specific spin labels were partitioned throughout the protoplast or retained in the plasmalemma of Avena sativa L. cv. Garry and Park. Many classes of spin label were tested, including phospholipids, fatty acid, fatty acid methyl ester, maleimide, iodoacetamide, short chain hydrocarbon, androstane, 2,2,6,6-tetramethyl-4-aminopiperidinooxyl (TEMPAMINE) and 2,2,6,6-tetramethylpiperidinooxyl (TEMPO). All except the phosphotidylcholine spin label were found to partition throughout the cell. The phosphotidylcholine spin label may have been selectively retained in the plasmalemma.

Journal Article↗
PubMedDate not supplied

[Macromolecule rotative correlation time measurement by ESR for covalently bound spin label].

The dependence from temperature and viscosity of the shifts of the internal and external wide extremums in the ESR spectra of spin labelled bovine serum albumin has been studied. 2,2,6,6-tetramethylpiperidine-NI-oxyl-4-iodacetamide was used as a spin label. The obtained dependences was shown to be a consequence of the label participation in two types of rotations: an anisotropic fast rotation with tau less than 10(-9) sec relatively to a macromolecule, and the isotropic one with tau greater than 10(-8) sec due to rotation of the macromolecule itself. These conclusions were done on the basis of a model for complex rotation of the spin label. Comparison of theoretical and experimental data makes it possible to determined the correlation time for the protein moiety, to evaluate quantitatively the polarity of surroundings of the iminoxyl and to introduce a numerical parameter for the degree of mobility of the spin label relatively to protein molecule.

Electron Spin Resonance Spectroscopy↗
PubMed2005

Dipole-dipole interactions in spin-labeled au nanoparticles as a measure of interspin distances.

A series of Au nanoparticles modified with a nitroxide-functionalized ligand was prepared with a range of spin-label coverage. The X-band EPR spectra of frozen solutions of these nanoparticles showed coverage-dependent line-broadening due to dipole-dipole interactions between spin labels. We developed a methodology to analyze such spectra in terms of geometrical features of the nanoparticles (e.g., Au core size and the length of the spin-labeled ligand). Our method is based on the assumption that the spectral line shape is determined by the average distance between nearest-neighboring spin labels adsorbed on the Au particle. Geometrical and statistical analysis then relates this distance to the line shape parameter d1/d, which was calibrated using a model system. Application of this methodology to the experimental spectra provided information about the conformation of ligands on the Au surface. We found that, if the spin-labeled ligand is substantially longer than the surrounding protecting layer, it does not adopt a fully stretched conformation but wraps around the particle immediately above the layer of surrounding ligand. Our results also show that the ligands do not adsorb cooperatively on the Au surface.

Journal Article↗
PubMedDate not supplied

Rotational correlation times of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy spin label with respect to heme and nonheme proteins.

Noncovalent spin labeled proteins (ovalbumin, bovine serum albumin, hemoglobin, and cytochrome c) were investigated in order to follow the different type of interactions between the nitroxide radical of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy spin label and functional groups of heme and nonheme proteins as well as the pH influence on molecular motion of the label with respect to these proteins. EPR spectra were recorded at room temperature and the computer simulation analysis of spectra was made in order to obtain the magnetic parameters. Noncovalent labeling of proteins can give valuable information on the magnetic interaction between the label molecule and the paramagnetic center of the proteins. The relevance of this interaction can be obtained from line shape analysis: computer simulations for nonheme proteins assume a Gaussian line shape, whereas for heme proteins, a weighted sum of Lorentzian and Gaussian components is assumed. In the framework of the "moderate jump diffusion" model for rotational diffusion, the rotational correlation time is strongly influenced by pH, because of the electrostatic interactions and hydrogen bonding.

Cyclic N-Oxides↗