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PubMed1999

Linewidth analysis of spin labels in liquids. II. Experimental.

This work demonstrates that homogeneous linewidths can be extracted from continuous wave electron paramagnetic resonance spectra and that they quantitatively agree with the predictions of existing relaxation theory. We suggest that relaxation theory can be used to predict experimental lineshapes provided that the simulations properly include sources of broadening. We have found that the rotational correlation times for spin labels in different percentages of glycerol/water mixtures are best modeled by a power law treatment for the viscosity, similar to that for translational diffusion. The translational diffusion coefficients themselves also have a power law dependence on the viscosity for glycerol/water mixtures. The linewidths were linearly dependent upon both the oxygen and the spin label concentration. The hyperfine splittings of all nuclei were observed to decrease linearly with increasing spin label concentration, completely at odds with existing theory which predicts a quadratic dependence upon concentration. The linear dependence was independent of hyperfine splitting until the magnitude of the hyperfine splitting was less than the homogeneous linewidth.

Computer Simulation↗
PubMed2005

Grading of CNS neoplasms using continuous arterial spin labeled perfusion MR imaging at 3 Tesla.

PURPOSE: To differentiate glioma grade based on blood flow measured using continuous arterial spin labeled (CASL) perfusion MRI, implemented at 3 Tesla for improved signal-to-noise ratio (SNR) and spin labeling effect. MATERIALS AND METHODS: CASL perfusion images were obtained preoperatively in 26 patients with brain neoplasms (19 high-grade gliomas (HGGs; WHO grades 3 and 4) and seven low-grade gliomas (LGGs; WHO grades 1 and 2)). The mean and maximum tumor blood flow (TBF and TBFmax) were calculated in the neoplasm, including surrounding infiltrating tumor vs. edema. Measures normalized to global CBF (nTBF and nTBFmax) were also obtained. RESULTS: Normalized measures of TBFmax provided the best distinction between HGG and LGG groups (Wilcoxon rank sum test, P = 0.01). Seventeen of 19 HGGs showed nTBFmax > 1.0, and 15 of 19 showed nTBFmax > 1.3. Four of seven LGGs showed nTBFmax < 1.0, and six of seven showed nTBFmax < 1.3. Absolute TBFmax also differed significantly between the HGG and LGG groups (P = 0.04). TBFmax in 11 of 17 HGGs was >50 mL/100 g/min (mean +/- SD = 94.9 +/- 71.7 mL/100 g/min). All but one LGGs showed TBFmax < or = 50 mL/100 g/min (mean +/- SD = 42.8 +/- 22.0 mL/100 g/min). CONCLUSION: CASL perfusion MRI provides a quantitative, noninvasive alternative to dynamic susceptibility contrast perfusion MR methods for evaluating gliomas.

Adult↗
PubMed2006

In vivo estimation of the flow-driven adiabatic inversion efficiency for continuous arterial spin labeling: a method using phase contrast magnetic resonance angiography.

The accurate quantification of perfusion with arterial spin labeling (ASL) requires consideration of a number of factors, including the efficiency of the inversion and control pulses used for spin labeling. In this study the effects of spin velocity on continuous ASL efficiency when using the amplitude modulated control strategy were investigated using simulations of the Bloch equations. The inversion efficiency was determined in vivo by combining the simulations with phase-contrast velocity mapping data acquired at the level of the tagging plane. Using this novel method, an average inversion efficiency of 69% was calculated for a group of 28 subjects, in good agreement with experimental data reported previously. There was, however, a large range in inversion efficiency measured across the subject group (50-76%), indicating that the velocity dependence of the amplitude modulated control efficiency may introduce additional variability into the perfusion calculations if not properly taken into account.

Algorithms↗
PubMed1994

Binding of fluorescent and spin-labeled C-terminal hirudin analogs to thrombin.

Synthetic peptides based on the sequence of the negatively charged carboxyl tail of hirudin exhibit anticoagulant activity. Several antithrombin agents are being developed by chemical and structural optimization of these "hirupeptides". The present work demonstrates the design and use of novel spin-labeled and fluorescent-labeled C-terminal hirudin analogs to study the interactions of these antithrombin agents with thrombin in solution. Three labeled hirulabels were synthesized based upon the amino acid sequence of the antithrombin agent MDL 28050, X-NH-(CH2)7-CO-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-Cha-D-Glu-OH, where X = anthraniloyl, 1,5-dansyl, or 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl. The modifications did not significantly alter the potency of these inhibitors which showed Ki values of 100 nM. Their interactions with human and bovine thrombin were studied by ESR and fluorescence techniques. The spin-labeled hirupeptide was able to discern subtle differences in binding to human versus bovine thrombin. The 8-aminooctanoic acid spacer arm placed the nitroxide moieties near the active site, near regions of the autolysis loops which differentiates between human alpha- and gamma-thrombin. It was also able to discern paramagnetic quenching and fluorescence energy transfer interactions, respectively, between covalently attached spin labels and fluorescent probes at the active site Ser 195 and the fluorophore on the hirupeptide.

Amino Acid Sequence↗
PubMed1988

Synthesis of a spin-labeled phospholipid for studying membrane dynamics in intact mammalian cells.

We report here the synthesis of a spin-labeled phospholipid, 1-palmitoyl-2-(4-doxylpentanoyl)glycerophosphocholine. The synthetic route for this probe involves two major steps: 1) the synthesis of 4-doxylpentanoic acid from ethyl levulinate and 2-amino-2-methyl propanol, and 2) the synthesis of the lipid from 4-doxylpentanoic acid and lysolecithin. The efficiency and yield of both steps have been greatly improved. This represents the first instance that the synthesis of this important spin-labeled phospholipid is described in detail. Because it mimics the native lipid molecule and can be readily incorporated into biological membranes, this probe should be extremely useful for studying lipid dynamics in the plasma membrane of intact mammalian cells using electron spin resonance (ESR) spectroscopy.

Animals↗
PubMed1998

Oxygen permeability of thylakoid membranes: electron paramagnetic resonance spin labeling study.

Oxygen transport in thylakoid membranes of spinach chloroplasts (Spinacia oleracea) has been studied by observing the collisions of molecular oxygen with spin labels, using line broadening electron paramagnetic resonance (EPR) spectroscopy. Stearic acid spin labels were used to probe the local oxygen diffusion-concentration product. The free radical moiety was located at various distances from the membrane surface, and collision rates were estimated from linewidths of the EPR spectra measured in the presence and absence of molecular oxygen. The profile of the local oxygen diffusion-concentration product across the membrane determined at 20 degrees C demonstrates that this product, at all membrane locations, is higher than the value measured in water. From the profile of the oxygen diffusion-concentration product, the membrane oxygen permeability coefficient has been estimated using the procedure developed earlier (W.K. Subczynski, J.S. Hyde, A. Kusumi, Proc. Natl. Acad. Sci. USA 86 (1989) 4474-4478). At 20 degrees C, the oxygen permeability coefficient for the lipid portion of the thylakoid membrane was found to be 39.5 cm s-1. This value is 20% higher than the oxygen permeability coefficient of a water layer of the same thickness as the thylakoid membrane. The high permeability coefficient implies that the oxygen concentration difference across the thylakoid membrane generated under the illumination of the leaf by saturating actinic light is negligible, smaller than 1 microM.

Cell Membrane Permeability↗
PubMed1979

The binding of spin-labeled derivatives of NAD+ and its structural components to pig skeletal muscle lactate dehydrogenase.

The binding of spin-labeled derivatives of NAD+ and its structural components to pig skeletal muscle lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) is described. In contrast to results previously obtained with the heart muscle isozyme (Wenzel, H.R., Pfleiderer, G., Trommer, W.E., Paschenda, K. and Redhardt, A. (1976) Biochim. Biophys. Acta 452,292--301), no significant increase is observed in the binding constant of N6-SL-ADP as compared N6-SL-AMP. This different behavior can be explained by the substitution of glutamine-31 for alanine in the muscle isozyme, which has been proposed to account for the the tighter binding of NADH to the heart type. In both isozymes the binding of the binding of the spin-labeled coenzyme itself is weaker than found for its structural components.

Animals↗
PubMed2006

Dynamic behavior of fatty acid spin labels within a binding site of soybean lipoxygenase-1.

The putative substrate-binding site in lipoxygenases is long and internal. There is little direct evidence about how the unsaturated fatty acid substrates enter and move within the cavity to position themselves correctly for electron transfer reactions with the catalytic non-heme iron. An EPR spectroscopy approach, with spin-labeled fatty acids, is taken here to investigate dynamic behavior of fatty acids bound to soybean lipoxygenase-1. The probes are labeled on C5, C8, C10, C12, and C16 of stearic acid. The EPR-determined affinity for the enzyme increases as the length of the alkyl end of the probe increases, with a DeltaDeltaG of -190 cal/methylene. The probes in the series exhibit similar enhanced paramagnetic relaxation by the iron center. These results indicate that the members of the series have a common binding site. All of the bound probes undergo considerable local mobility. The stearate spin-labeled at C5 has the highest affinity for the lipoxygenase, and it is a competitive inhibitor, with a K(i) of 9 muM. Surprisingly, this stearate labeled near the carboxyl end undergoes more local motion than those labeled in the middle of the chain, when it is bound. This shows that the carboxyl end of the fatty-acid spin label is not rigidly docked on the protein. During catalysis, repositioning of the substrate carboxyl on the protein surface may be coupled to motion of portions of the chain undergoing reaction.

Catalytic Domain↗
PubMed2000

Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data.

To test whether distances derived from paramagnetic broadening of (15)N heteronuclear single quantum coherence (HSQC) resonances could be used to determine the global fold of a large, perdeuterated protein, we used site-directed spin-labeling of 5 amino acids on the surface of (15)N-labeled eukaryotic translation initiation factor 4E (eIF4E). eIF4E is a 25 kDa translation initiation protein, whose solution structure was previously solved in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) micelle of total molecular mass approximately 45-50 kDa. Distance-dependent line broadening consistent with the three-dimensional structure of eIF4E was observed for all spin-label substitutions. The paramagnetic broadening effects (PBEs) were converted into distances for modeling by a simple method comparing peak heights in (15)N-HSQC spectra before and after reduction of the nitroxide spin label with ascorbic acid. The PBEs, in combination with HN-HN nuclear Overhauser effects (NOEs) and chemical shift index (CSI) angle restraints, correctly determined the global fold of eIF4E with a backbone precision of 2.3 A (1.7 A for secondary structure elements). The global fold was not correctly determined with the HN-HN NOEs and CSI angles alone. The combination of PBEs with simulated restraints from another nuclear magnetic resonance (NMR) method for global fold determination of large proteins (methyl-protonated, highly deuterated samples) improved the quality of calculated structures. In addition, the combination of the two methods simulated from a crystal structure of an all alpha-helical protein (40 kDa farnesyl diphoshphate synthase) correctly determined the global fold where neither method individually was successful. These results show the potential feasibility of obtaining medium-resolution structures for proteins in the 40-100 kDa range via NMR.

Amino Acid Sequence↗
PubMed1989

Polydispersity of aggregates formed by the polyene antibiotic amphotericin B and deoxycholate. A spin label study.

The amphotericin B-deoxycholate (AB-DOC) system (1:2, mole basis) was studied with regard to its organizational properties making use of spin label ESR spectra. The spectra of a fatty acid spin label intercalated in AB-DOC preparations revealed two components, one strongly (S) and one weakly (W) immobilized. Spectral subtractions indicated that S corresponds to label in mixed AB-DOC aggregates while W is due to label in deoxycholate micelles. This situation, coexistence of different aggregates, is similar to that found in systems consisting of bile salts and phospholipids. The DOC/AB mole ratio in the mixed aggregate is highest when pure DOC micelles are present. Dilution leads to disappearance of the latter and to continuous loss of DOC from AB-DOC accompanied by an increase in size and decrease in solubility of the aggregates, as verified by filtration and centrifugation experiments. The results indicate that AB-DOC systems are polydisperse. Since amphotericin B preparations having different organizational properties display different toxic and therapeutic effect, the study of amphotericin B aggregates should help in understanding these phenomena at a molecular level.

Amphotericin B↗
PubMed1983

Studies of the mobility of maleimide spin labels within the erythrocyte membrane.

We have confirmed a method yielding reproducible and reliable spectrometric parameters derived from spin-labeled erythrocyte ghosts using nitroxide derivatives of maleimide compounds. The disorder parameter, W/S, was shown to vary with changes in the structure of the label, the conditions utilized for labeling such as ionic strength and erythrocyte age and the presence of drugs such as alcohol and acetaminophen. The nitroxide spectrum was also found to change with increasing and decreasing temperature in an irreversible manner. These findings should permit increased reliance to be placed on the spin-labeling technique when used to monitor changes in membrane lipid or protein assembly.

Cyclic N-Oxides↗
PubMed1998

High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes.

The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the sn-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic 14N-hyperfine splittings are resolved in the gzz and gyy regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged element of the 14N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the 14N-hyperfine manifolds in the gzz region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.

Anisotropy↗
PubMedDate not supplied

[Interaction of spin-labeled analogues of vitamin B 6 with the active site of apotransaminase].

Spin-labeled analogues of vitamin B6: 2, 2, 6, 6-tetramethyl-N-oxylpiperydinyl-4-(5' phosphopyridoxyl)-amine (1) and 2, 2, 6, 6-tetramethyl-N-oxyl-piperydinyl-4-(pyridoxal-5')-phosphate (II) are synthesized. There analogues were shown to interact in the equimolar ratio with the active site of cytosol aspartate transaminase. It was proved by CD-titration of apotransaminase with I and II and by competition between the coenzyme and synthesized analogues. The free valency of spin-labeled coenzymes immediately disappears after interaction with the apoenzyme due to iminoxyl group reduction. The binding of I and II with the apoenzyme is accompanied by oxidation of one of the inner cysteine residues. The reactivation of the modified apoenzyme with PLP is not less than 65% of original transaminase activity. The analysis of space-filling atomic models of synthesized compounds allows to conclude that the distance between the centre of pyridine ring of the coenzyme and the modified thiol group is not more than 8 A.

Apoproteins↗
PubMed1993

Exploration of the single-stranded DNA-binding domains of the gene V proteins encoded by the filamentous bacteriophages IKe and M13 by means of spin-labeled oligonucleotide and lanthanide-chelate complexes.

Scrutiny of NOE data available for the protein encoded by gene V of the filamentous phage IKe (IKe GVP), resulted in the elucidation of a beta-sheet structure which is partly five stranded. The DNA-binding domain of IKe GVP was investigated using a spin-labeled deoxytrinucleotide. The paramagnetic-relaxation effects observed in the 1H-NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized using two-dimensional difference spectroscopy. In this way, the residues present in the DNA-binding domain of IKe GVP can be located in the structure of the protein. They exhibit a high degree of identity with residues in the gene V protein encoded by the distantly related phage M13 (M13 GVP), for which similar spectral perturbations are induced by such a spin-labeled oligonucleotide. Binding studies with negatively charged lanthanide-1,4,7,10-tetraazacyclodecanetrayl-1,4,7-10- tetrakis(methylene)tetrakisphosphonic acid (DOTP) complexes, showed that these complexes bind to IKe and M13 GVP at two spatially remote sites whose affinities have different pH dependencies. Above pH 7, there is one high-affinity binding site for Gd(DOTP)5-/M13 GVP monomer, which coincides with the single-stranded DNA-binding domain as mapped with the aid of spin-labeled oligonucleotide fragments. The results show that single-stranded DNA binds to conserved (phosphate binding) electropositive clusters at the surface of M13 and IKe GVP. These positive patches are interspersed with conserved or conservatively replaced hydrophobic residues. At pH 5, a second Gd(DOTP)(5-)-binding site becomes apparent. The corresponding pattern of spectral perturbations indicates the accommodation of patches of conserved, or conservatively replaced, hydrophobic residues in the cores of the M13 and IKe dimers.

Amino Acid Sequence↗
PubMed1982

Cholesterol-phospholipid interaction in membranes. 1. Cholestane spin-label studies of phase behavior of cholesterol-phospholipid liposomes.

The effect of cholesterol concentration on the thermotropic phase behavior of aqueous phospholipid multi-bilayers was monitored by means of electron spin resonance spectroscopy (ESR) of a cholestane spin-label (CSL). The spin-label itself induces an additional transition in several different phospholipids, which is attributed to local melting around the spin probe. In contrast, cholesterol prevents its neighboring phospholipids from undergoing fluidization. Small additions of cholesterol affect the position of the probe-induced lipid mobilization curve. The phospholipid main gel-liquid-crystal transition, which is also observed as a separate change in probe mobilization, is not affected by low concentrations of cholesterol. These observations indicate the presence of two phases, a cholesterol-rich phase and a pure phospholipid phase, and indicate that CSL preferentially enters the cholesterol-rich phase. Addition of more than 20 mol % cholesterol abolishes the bulk phospholipid phase. This is evidenced by the disappearance of the gel-liquid-crystal transition as observed by ESR. However, the CSL-induced transition is present at all concentrations of cholesterol and CSL. The behavioral differences between the two sterols caution against using this probe as a direct substitute for cholesterol. However, it remains a useful tool for monitoring the phase behavior of cholesterol-phospholipid bilayer systems.

Cholestanes↗
PubMed2002

Myristoylated alanine-rich C kinase substrate (MARCKS) sequesters spin-labeled phosphatidylinositol 4,5-bisphosphate in lipid bilayers.

The myristoylated alanine-rich protein kinase C substrate (MARCKS) may function to sequester phosphoinositides within the plane of the bilayer. To characterize this interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), a novel spin-labeled derivative, proxyl-PIP(2), was synthesized and characterized. In the presence of molecules known to bind PI(4,5)P(2) the EPR spectrum of this label exhibits an increase in line width because of a decrease in label dynamics, and titration of this probe with neomycin yields the expected 1:1 stoichiometry. Thus, this probe can be used to quantitate the interactions made by the PI(4,5)P(2) head group within the bilayer. In the presence of a peptide comprising the effector domain of MARCKS the EPR spectrum broadens, but the changes in line shape are modulated by both changes in label correlation time and spin-spin interactions. This result indicates that at least some proxyl-PIP(2) are in close proximity when bound to MARCKS and that MARCKS associates with multiple PI(4,5)P(2) molecules. Titration of the proxyl-PIP(2) EPR signal by the MARCKS-derived peptide also suggests that multiple PI(4,5)P(2) molecules interact with MARCKS. Site-directed spin labeling of this peptide shows that the position and conformation of this protein segment at the membrane interface are not altered significantly by binding to PI(4,5)P(2). These data are consistent with the hypothesis that MARCKS functions to sequester multiple PI(4,5)P(2) molecules within the plane of the membrane as a result of interactions that are driven by electrostatic forces.

Calcium-Binding Proteins↗
PubMed1998

Spin-label studies of lipid-protein interactions with reconstituted band 3, the human erythrocyte chloride-bicarbonate exchanger.

Lipid-protein interactions in reconstituted band 3 preparations were investigated by using spin-labeled lipids in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Purified erythrocyte band 3 was reconstituted into egg phosphatidylcholine liposomes at high protein density with preservation predominantly of the dimeric state. Lipid-protein associations were revealed by the presence of a component in the EPR spectra that, when compared to spectra obtained from protein-free bilayers, indicated that lipid chain motions are restricted by interactions with the protein. From the fraction of the motionally restricted component obtained from the phosphatidylcholine spin-label, a value of 64 +/- 14 annular lipids per band 3 dimer was obtained. This agrees with a value of 62 for the number of lipids that may be accommodated around the electron density map of a band 3 dimer. Selectivity of various spin-labeled lipids for the protein revealed that androstanol had a lower affinity for the band 3 interface, whereas a distinct preference was observed for the negatively charged lipids phosphatidylglycerol and stearic acid over phosphatidylcholine. This preference for negatively charged lipids could not be screened by 1-M salt, indicating that electrostatic lipid-protein interactions are not dominant. Estimates of annular lipid exchange rates from measured acyl chain segmental motions suggested that the rate of exchange between bilayer and boundary lipids was approximately 10(6) s(-1), at least an order of magnitude slower than the rate of lipid lateral diffusion in protein-free bilayers.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1992

Irradiation increases proteolysis in erythrocyte ghosts: a spin label study.

X- and gamma-irradiation of human erythrocyte membranes (250-1000 Gy) was found to decrease the ratio of weakly to strongly immobilized signal height of membrane-bound maleimide spin label (Mal-6). Subsequent incubation of spin-labeled membranes at ambient temperature (21 degrees C) induced a progressive increase in this ratio, faster for membranes irradiated with low doses which was hampered by protease inhibitors. These results demonstrate that ionizing radiation stimulates proteolysis of erythrocyte membrane proteins by membrane-associated proteases.

Blood Proteins↗