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PubMed1975

Proton nuclear magnetic resonance of spin-labeled Escherichia coli tRNAf1MET.

Thiouridine at position 8 (s4U8) of tRNAf1Met was spin-labeled with the nitroxide free radical, N-(1-oxyl-2,2,5,5-Tetramethyl-3-pyrrolidinyl) bromacetamide, for proton nuclear magnetic resonance spectroscopic studies. The well-resolved methyl peak of ribothymidine is unperturbed, but the peak tentatively assigned to the C-5 methylene group of dihydrouridine is considerably broadened in spin-labeled tRNAf1Met. Of the approximately 27 slowly exchanging protons observed in the region between 11 and 15 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonic acid, the equivalent of about five protons apparently disappeared in spin-labeled tRNAf1Met. The well-resolved single proton at 14.8 ppm was missing not only in the paramagnetic species, but also in the diamagnetic reduced form of spin-labeled tRNAf1Met, and was unequivocally identified as a hydrogen bond involving s4U8 by comparison of several forms of tRNAf1Met specifically modified at s4U. Evidence that the perturbation of a second single proton resonance at 14.6 ppm (shift and broadening) is coupled to the loss of a tertiary hydrogen bond involving residue 8, arises from the same modified forms. The resolved resonances in the methyl and N-H regions, particularly the resonance at 14.6 ppm as well as the four N-bonded proton resonances at higher field which are broadened solely due to their proximity to the unpaired electron of the spin label, provide specific indicators of the geometry of tRNAf1Met structure in solution. Their observability by nuclear magnetic resonance spectroscopy opens up the possibility of monitoring distance changes among the base residues of spin-labeled tRNAf1Met upon its interaction with aminoacyl-tRNA synthetase and other enzymes.

Binding Sites↗

PubMed1988

Conformational change in thrombospondin induced by removal of bound Ca2+. A spin label approach.

The effect of removal of Ca2+ bound to thrombospondin (TSP) on the protein structure in solution has been investigated using ESR spin-label techniques. A maleimide spin label was selectively attached to the free thiol group presumably near the carboxyl-terminal domain in which Ca2+-binding sites are situated. The ESR spectra of spin-labeled TSP showed that the bound label undergoes a relatively fast rotational motion with an effective rotational correlation time in the nano-second time regimes. Removal of bound Ca2+ in TSP by dialyzing spin-labeled TSP from a Ca2+-containing buffer into an EDTA-containing buffer resulted in an increase in the mobility of the bound label by a factor of 2.3. The data suggest that EDTA chelation of bound Ca2+ in TSP induces a conformational change of TSP at least near the site of spin labeling.

Blood Platelets↗

PubMedDate not supplied

[Spin-spin interaction upon introduction of a spin label into immunoglobulins M and G at the carbohydrate moiety].

By spin labeling the monoclonal IgM and normal IgG at the carbohydrate moiety with 2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl, preparations were obtained whose ESR spectra indicate rapid exchange spin-spin interactions between two spin labels. It was shown that, in the case of spin-labeled IgM, this spectrum is determined by a glycopeptide noncovalently bound to IgM, which incorporates two spin labels.

Chromatography, Gel↗

PubMed1976

Binding of a metyrapone spin label to microsomal cytochrome P-450.

Probing of the active site of microsomal cytochrome P-450 was carried out with a spin label derived from 2-methyl-1,2-bis(3-pyridyl)-1-propanone (metyrapone). Its optical binding spectra to cytochrome P-450 resemble the spectra with metyrapone. The electron paramagnetic resonance spectrum of the spin label in microsomes indicates binding with strong immobilization. Since the apparent optical and EPR binding constants agree very well (Ks approximately 2-10(-5) M), and metyrapone is found to displace the spin label, we conclude, that the spin label binds to the active site of cytochrome P-450. Addition of detergents or an increase in temperature mobilizes the bound spin label slightly. The EPR signal accounts for only 60% of the bound spin label due to its dipolar interaction with the low-spin ferric heme of cytochrome P-450. From this finding, the distance between nitroxide and iron is evaluated to be 11 A. This supports the model that one of the pyridine nitrogens of metyrapone is coordinated to the iron of cytochrome P-450. The bound spin label shows virtually no interaction with ferricyanide as if the active site of cytochrome P-450 is a hydrophobic pocket not accessible to ions.

Animals↗

PubMed1977

Fatty acid enhancement of human serum albumin binding properties. A spin label study.

The introduction of a new spin-labeled anionic ligand, 1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene, is reported. Under the experimental conditions, the first molar equivalent of this ligand is 93% bound to human serum albumin. With the addition of palmitate, the free spin label concentration decreases greatly, by almost 80%, in the presence of a fatty acid:albumin ratio of 3:1 to 4:1. The spectral characteristics of the bound spin label are also affected. The changes seen in the intensity of and the splitting between the high and low field extrema are indicative of perturbations of the protein molecule. It is seen then that the binding of each molar equivalent of fatty acid effects the conformation state of albumin and allosterically affects albumin binding properties. Computer spectral subtractions, furthermore, suggest that the binding of the first molar equivalent of palmitate specifically increases the affinity of the first two 1-gamma-amino-butyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene binding sites. The present results indicate that fluctuations in serum free fatty acid levels within the physiological range may have a major modulatory effect on the free serum levels of certain drugs and/or physiological substances that bind to albumin.

Binding Sites↗

PubMed1996

Effects of lutein and cholesterol on alkyl chain bending in lipid bilayers: a pulse electron spin resonance spin labeling study.

A short pulse saturation recovery electron spin resonance technique has been used to study the effects of polar carotenoid-lutein and cholesterol on interactions of 14N:15N stearic acid spin-label pairs in fluid-phase phosphatidylcholine (PC) membranes. Bimolecular collisions for pairs consisting of various combinations of [14N]-16-, [14N]-10-, [14N]-7-, or [14N]-5-doxylstearate and [15N]-16-doxylstearate in dimyristoyl-PC (DMPC) or egg yolk PC (EYPC) membranes were measured at 27 degrees C. In the absence and presence of lutein or cholesterol for both lipid systems, the collision rates were ordered as 16:5 < 16:7 < 16:10 < 16:16. For all spin-label pairs studied, interaction frequencies were greater in DMPC than in EYPC. Polar carotenoid-lutein reduces the collision frequency for all spin-label pairs, whereas cholesterol reduces the collision frequency for 16:5 and 16:7 pairs and increases the collision frequency in the membrane center for 16:10 and 16:16 pairs. The presence of unsaturated alkyl chains greatly reduces the effect of lutein but magnifies the effect of cholesterol in the membrane center. The observed differences in the effects of these modifiers on alkyl chain bending result from differences in the structure of cholesterol and polar carotenoid and from their different localization within the lipid bilayer membrane. These studies further confirm the occurrence of vertical fluctuations of alkyl chain ends toward the bilayer surface.

Cholesterol↗

PubMed2005

Four-phase single-capillary stepwise model for kinetics in arterial spin labeling MRI.

An extended model for extracting measures of brain perfusion from pulsed arterial spin labeling (ASL) data while considering transit effects and restricted permeability of capillaries to blood water is proposed. We divided the time course of the signal difference between control and labeled images into four phases with respect to the arrival time of labeled blood water at the voxel of interest (t(A)), transit time through the arteries in the voxel (t(ex)), and duration of the bolus of labeled spins (tau). Dividing the labeled slab of blood water into many discrete segments, and adapting numerical integration methods allowed us to conveniently model restricted capillary-tissue exchange based on a modified distributed parameter model. We compared this four-phase single-capillary stepwise (FPSCS) model with models that treat water as a freely diffusible tracer, using both simulations and experimental ASL brain imaging data at 1.5T from eight healthy subjects (24-80 years old). The FPSCS model yielded less errors in the least-squares sense in fitting brain ASL data in comparison with freely diffusible tracer models of water (P = 0.055). These results imply that restricted permeability of capillaries to water should be considered when brain ASL data are analyzed.

Adult↗

PubMed1991

Al3+ versus Ca2+ ion binding to methionine and tyrosine spin-labeled bovine brain calmodulin.

Bovine calmodulin analogues, spin-labeled at either methionine or tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies to investigate possible calmodulin interactions with aluminum ion. The study attempts to clarify a previous report in the literature (H. Siegel, R. Coughlin, and A. Haug, Biochem. Biophys. Res. Commun. 115, 512 (1983)) which indicated, on the basis of EPR experiments on methionine spin-labeled protein, significant interaction between calmodulin and aluminum ion at pH = 6.5. In EPR metal ion titration experiments we have found that the signal line-shape (from both methionine and tyrosine spin labels) changed dramatically with the addition of calcium ion, but was virtually unchanged with the addition of aluminum ion at pH = 6.5. Experiments performed at pH = 5.5, where significantly more "free" aluminum ion (i.e., Al(H2O)6(3+) = Al3+) is present, also failed to produce the line-narrowing effect observed in the earlier study. Based on our EPR experiments, in the pH range 5.5 to 6.5, we find no evidence for significant interaction between calmodulin and aluminum ion.

Aluminum↗

PubMed2004

[Noninvasive measurement of relative cerebral blood flow with the blood bolus MRI arterial spin labeling: basic physics and clinical applications].

Knowledge of tumor blood flow is important for diagnosis and follow-up of brain tumors after therapy, especially to discriminate necrosis from tumor recurrence after radiation or chemotherapy. Meanwhile, perfusion and diffusion MRI, besides MR-angiography, are state of the art in stroke imaging. Until now, perfusion imaging was mostly performed using the first-pass dynamic susceptibility-weighted contrast-enhanced (DSC) MRI. The MRI-based arterial spin labeling technique (ASL) is a novel approach for measuring relative cerebral blood flow (rCBF) without using extrinsic contrast agents, by labeling spins of flowing arterial blood as intrinsic contrast agent. This article describes physical basics of ASL and shows clinical examples in neuroimaging such as in meningeoma, glioblastoma, oligodendroglioma, and cerebral ischemia, using the Q2TIPS ASL technique. Gray matter is clearly visible, while the observed white matter signal obtained by Q2TIPS is only slightly higher than background noise. Venous blood causes artefacts in the sagittal sinus and other large superficial veins in the subarachnoid space. Meningeoma and glioblastoma show elevated rCBF, whereas oligodendroglioma and cerebral ischemia have reduced rCBF values. Arterial-spin-labeling techniques are noninvasive tools for measuring rCBF within 5 min, using a standard MRI scanner.

Blood Flow Velocity↗

PubMed1983

Interaction of concanavalin A with spin-labeled glycolipid incorporated into liposomes.

Using a synthetic glycolipid derived from maltotetraose and a spin-labeled fatty acid, the lateral distribution and molecular motion of the spin-labeled glycolipid on phosphatidylcholine liposomes in the presence and absence of concanavalin A were examined. When the spin-labeled glycolipid was added to preformed egg yolk phosphatidylcholine-dicetyl phosphate (molar ratio, 10 : 1) liposomes, most of the spin-labeled glycolipid molecules could be incorporated into liposomes as shown by their concanavalin A-induced agglutination. Concanavalin A also caused a change in line width of the ESR signal of liposome-bound spin-labeled glycolipid, whereas the overall splitting value 2A parallel did not change significantly. It is suggested that the binding of glycolipid molecules to concanavalin A increased the interactions among the radicals of the probe but that the mobility of the acyl chain off glycolipids was not affected. These signal changes were also observed with succinyl-concanavalin A. However, in contrast to concanavalin A, no appreciable agglutination of liposomes could be induced by the latter concanavalin A derivative. Both the agglutination of liposomes and the change in line width of the ESR signal were completely inhibited by alpha-methyl-D-mannoside.

Agglutination Tests↗

PubMed1971

Structural measurements in hemoprotiens: use of spin-labeled protoheme as a probe of heme environment.

With the aid of two kinds of spin-labeled protohemins, the nature of the heme-protein interaction of various hemoproteins was investigated. Di- and mono-spin-labeled protohemins were prepared from protohemin and 2,2,5,5-tetramethyl-3-aminopyrrolidine-1-oxyl. The spin-labeled hemins were recombined with apoproteins of hemoglobin (Hb), myoglobin (Mb), cytochrome c peroxidase (EC 1.11.1.5) and horseradish peroxidase (EC 1.11.1.7). Electron paramagnetic resonance spectra of the di- and mono-spin-labeled hemoglobin in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees C exhibited moderate immobilization of the labels, while that of cytochrome c peroxidase showed stronger immobilization. Di-spin-labeled horseradish peroxidase showed an EPR spectrum of a simple broad line with peak-to-peak line width of 35 G. This broadening is due to spin-spin interaction between the two labels attached at the 6- and 7-positions of the porphyrin ring. Ligand binding to the spin-labeled hemoproteins altered the EPR line shapes and amplitudes. The former is attributed to the changes in the mobility of the labels and the latter to the magnetic dipolar interaction between the heme iron and free radical. From the strength of this interaction the distance between the iron and the nitroxide radical may be calculated. In the hemoproteins examined, the distances are: Hb 12.5 A, Mb 12.0 A, cytochrome peroxidase approximately 14 A, and horseradish peroxidase 9.0 A.

Chromatography↗

PubMed2000

High-field EPR studies of the structure and conformational changes of site-directed spin labeled bacteriorhodopsin.

Cw and pulsed high-field EPR (95 GHz, 3.4 T) are performed on site-directed spin labeled bacteriorhodopsin (BR) mutants. The enhanced Zeeman splitting leads to spectra with resolved g-tensor components of the nitroxide spin label. The g(xx) component shift determined for 10 spin labels located in the cytoplasmic loop region and in the protein interior along the BR proton channel reveals a maximum close to position 46 between the proton donor D96 and the retinal. A plot of g(xx) versus A(zz) of the nitrogen discloses grouping of 12 spin labeled sites in protic and aprotic sites. Spin labels at positions 46, 167 and 171 show the aprotic character of the cytoplasmic moiety of the proton channel whereas nitroxides at positions 53, 194 and 129 reveal the protic environment in the extracellular channel. The enhanced sensitivity of high-field EPR with respect to anisotropic reorientational motion of nitroxides allows the characterization of different motional modes for spin labels bound to positions 167 and 170. The motional restriction of the nitroxide at position 167 of the double mutant V167C/D96N is decreased in the M(N) photo-intermediate. An outward shift of the cytoplasmic moiety of helix F in the M(N) intermediate would account for the high-field EPR results and is in agreement with diffraction and recent X-band EPR data.

Bacteriorhodopsins↗

PubMed2006

Characterization of the linker 2 region in human vimentin using site-directed spin labeling and electron paramagnetic resonance.

Site-directed spin labeling and electron paramagnetic resonance were used to probe residues 281-304 of human vimentin, a region that has been predicted to be a non-alpha-helical linker and the beginning of coiled-coil domain 2B. Though no direct test of linker structure has ever been made, this region has been hypothesized to be flexible with the polypeptide chains looping away from one another. EPR analysis of spin-labeled mutants indicates that (a) several residues reside in close proximity, suggesting that adjacent linker regions in a dimer run in parallel, and that (b) the polypeptide backbone is relatively rigid and inflexible in this region. However, this region does not show the characteristics of a coiled-coil as has been identified elsewhere in the molecule. Within this region, spectra from positions 283 and 291 are unique from all others thus far examined. These positions, predicted to be in a noncoiled-coil structure, display a significantly stronger interaction than the a-d contact positions of coiled-coil regions. Analysis of the early stages of assembly by dialysis from 8 M urea and progressive thermal denaturation shows the close apposition and structural rigidity at residues 283 and 291 occurs very early in assembly and with a relatively sudden onset, well before coiled-coil formation in other parts of the molecule. These features are inconsistent with hypotheses that envision the linkers as flexible regions, or as looping away from one another, and raise the possibility that the linker may be the site at which dimer alignment and/or formation is initiated. Spin labels placed further downstream yield spectra suggesting that the first regular heptad of rod domain 2 begins at position 302. In conjunction with our previous characterization of region 305-336 and the solved structure of rod 2B from 328-405, the full extent of coiled-coil domain in rod 2B is now known, spanning from vimentin positions 302-405.

Electron Spin Resonance Spectroscopy↗

PubMed1979

Identification of four major classes of sulfhydryl groups in human blood platelets. Ferricyanide titration of spin-labeled platelets.

Human blood platelets have been labeled with the sulfhydryl-specific spin labels, 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl and 3-maleimido-2,2,5,5-tetramethylpyrrolidine-1-oxyl. First, the ESR spectra of platelets labeled with either reagent revealed two classes of sulfhydryl groups, a mobile class and an immobile class. Second, when spin-labeled platelets were titrated with high concentrations of potassium ferricyanide (greater than 10(-3) M), there was a decrease in the peak heights of the mobile class of sulfhydryl groups due to dipole-dipole exchange. Third, plots of peak heights of the mobile class versus ferricyanide concentration revealed three classes of mobile sulfhydryl groups compared to a single immobile class. This technique may be used to show the relative locations of spin-labeled groups on cell surfaces.

Blood Platelets↗

PubMed1975

A spin-label study on fusion of red blood cells induced by hemagglutinating virus of Japan.

Fusion of red blood cells (RBC) induced by hemagglutinating virus of Japan (HVJ) has been studied using a phosphatidylcholine spin label. The spin label was readily incorporated and diffused into the lipid bilayer portion of the viral envelope. The exchange broadening in the electron spin resonance (ESR) spectrum of densely labeled virus disappeared rapidly when the virus was mixed with RBC at 37 degrees. The spectrum gradually approached that of the host cell spin labeled with the phosphatidylcholine label. The results directly indicate transfer and intermixing of phospholipid molecules between the viral envelope and RBC membrane. The transfer reaction was strongly dependent on temperature. No transfer was observed at lower temperatures where the virus adsorbed to the cell and caused aggregation but no hemolysis and fusion. The transfer rate remained negligibly small until 19 degrees and increased rapidly between 25 and 30 degrees. The virus-induced hemolysis showed similar temperature dependence. The transfer rate was greatly reduced under inhibitory conditions of fusion: glutaraldehyde treatment of RBC, trypsin treatment of HVJ, or the presence of concanavalin A. Only slight transfer was observed from fusion-inactive influenza virus to RBC. The transfer was greatly enhanced by the help of HVJ. The close parallelism suggests that the transfer and intermixing are necessary steps to the cell fusion. The transfer rate was dependent on fluidity of the host cell membrane and independent of the viral dose. The virus-induced transfer of phospholipid molecules between RBC's was also detected by the spin label. Its temperature dependence was quite similar to that for the virus-to-cell transfer. The intercellular transfer was nearly proportional to the viral dose.

Cell Fusion↗

PubMed1986

Lipid-protein interactions in cytochrome c oxidase. A comparison of covalently attached phospholipid photo-spin-label with label free to diffuse in the bilayer.

The aim of this study was to clarify the possible origins of the motion-restricted electron spin resonance (ESR) spectral component observed in membranes. For this purpose, a phospholipid photo-spin-label was synthesized, characterized, and used to study lipid-protein interactions in beef heart cytochrome c oxidase. The probe was designed with a nitroaryl azide incorporated in the phospholipid head group, and a spin-label on the sn-2 side chain, and was radiolabeled. The resulting molecule, 1-palmitoyl-2-(14-proxyl [2-3H]stearoyl)-sn-glycero-3-phospho-N-(4-azido-3-nitrophenyl)ethanolami ne, was stable under subdued light and during the procedures required to reconstitute cytochrome c oxidase in phospholipid bilayers. Upon photolysis, the photo-spin-label reacted with the protein in high yields (50% attached). There was no detectable destruction of the spin-label. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytochrome c oxidase after reaction with the photo-spin-label showed highest levels of attachment to bands I, III, and VII, with some labeling of other bands. The labeling pattern demonstrated a distribution of attachment sites, which was needed for the spin-labeling studies. ESR spectra of the attached labels at 25 degrees C indicated a constant fraction of motion-restricted lipid chains, independent of the lipid to protein ratio. In contrast, a spin-labeled phosphatidylcholine and the prephotolyzed photo-spin-label, both free to diffuse in the bilayer, exhibited behavior in agreement with the multiple equilibria binding model. These results, as well as data obtained with membranes frozen at -196 degrees C, show how several situations that lead to a motion-restricted ESR line shape can be distinguished. This study provides additional evidence that the fraction of lipids normally in contact with protein, and not aggregation artifacts, accounts for the observed motion-restricted component of ESR spectra of reconstituted cytochrome c oxidase in phospholipid bilayers.

Animals↗

PubMed2001

EPR studies of iso-1-cytochrome c: effect of temperature on two-component spectra of spin label attached to cysteine at positions 102 and 47.

Wild-type iso-1-cytochrome c from Saccharomyces cerevisiae containing naturally occurring cysteine at position 102 and mutated protein S47C (derived from the protein in which C102 had been replaced by threonine) were labeled with cysteine-specific methanethiosulfonate spin label. Continuous wave (CW) electron paramagnetic resonance (EPR) was used to examine the effect of temperature on the behavior of the spin label in the oxidized and reduced forms of wild-type cytochrome c and in the oxidized form of the mutated protein. The computer simulations revealed that the CW EPR spectrum for each form of cytochrome c consists of at least two components [a fast (F) and a slow (S) component], which differ in the values of the rotational correlation times tauRparallel (longitudinal rotational correlation time) and tauRperpendicular (transverse rotational correlation time) and that the relative contributions of the F and S components of the spectra change with temperature. In addition, the values of the rotational correlation times (tauRparallel and tauRperpendicular) for the F component appear to change much more dramatically with the temperature than the respective values for the S component. A large difference between the behavior of the oxidized and reduced wild-type spin-labeled cytochromes c indicates that the temperature-induced unfolding of the protein in the region around C102 progresses more rapidly when cytochrome c is in the oxidized form.

Cysteine↗

PubMed2004

Spin-labelled Au nanoparticles.

A series of Au nanoparticles functionalised with nitroxide spin labels has been prepared and studied by EPR spectroscopy. Samples with low coverage of the spin label were used to investigate the dynamics of the surface-attached labels at different distances from the Au surface. The rotational correlation times of spin labels vary from 10(-10) s to more than 3 x 10(-9) s, depending on the chain length of the label and the surrounding ligand. The samples with higher coverage of the spin label show an increasing contribution of the exchange interaction between nitroxides adsorbed in a close proximity to each other on the same nanoparticle. Quantitative analysis of the EPR spectra of these samples suggests the presence of non-equivalent binding sites on the surface of Au nanoparticles. Additionally, EPR signals of isolated radical pairs were observed at intermediate coverage.

Journal Article↗