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[Temperature dependence of the epr spectra of a spin-labeled myosin and its complex with adp].

The effect of temperature on ESR spectra of spin-labelled myosin and myosin-ADP complex was studied. It was shown that mobility of the sptrongly immobilized spin label almost did not change with temperature (-2 degrees C-37 degrees C) whereas mobility of the slightly immobilized spin label changed in a complicated manner and not monotonously. The observed changes in mobility of the slightly immobilized spin label and characteristics of the ESP spectrum of the myosin-ADP complex suggest that temperature causes continuous changes of the structure in the local environment of the binding sites of the spin label, and on the average, one conformation of myosin and myosin-ADP complex molecules exists at any temperature.

Adenosine Diphosphate↗
PubMed1986

Binding of N-acetylgalactosamine-specific lectins to spin-labeled galactosamine derivatives.

Legume seed lectins specific for N-acetyl-alpha-D-galactosaminyl end groups from Amphicarpaea bracteata, lima bean, Griffonia simplicifolia, Dolichos biflorus, and soybean were compared with respect to binding of several spin-labeled derivatives of D-galactosamine by electron spin resonance and precipitin inhibition analysis. Spin-label II [methyl 2-[[(2,2,5,5-tetramethyl-1-oxopyrrolidin-3-yl) carbonyl]amino]-2-deoxy-alpha-D-galactopyranoside], spin-label III [1-(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)-3-(2,2,6, 6-tetramethyl-1-oxypiperidin-4-yl)-2-thiourea], and spin-label IV [1-[4-[[(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)amino]carbonyl]phenyl]-3-(2, 2,6-tetramethyl-1-oxypiperidin-4-yl)-2-thiourea] contain 2-N-(oxypiperidinyl) or 2-N-(oxypyrrolidinyl) substituents varying in length and polarity of the linker arm between the glycoside and nitroxide ring. Spin-labels II and III were found to bind very weakly to all the lectins tested (Kd greater than or equal to 1.0 mM). Spin-label IV, containing a planar, nonpolar 2-N-phenyl group, was bound very strongly (Kd = 0.1-0.4 mM) and was moderately immobilized (2T parallel = 48-56 G) by all lectins except that from D. biflorus. Notably, the affinity of spin-label IV to lima bean lectin was 18-fold greater than that for methyl N-acetyl-alpha-galactosaminide. These results suggest that when the bulky oxypiperidinyl moiety lies in a position close to the sugar ring, it interferes with binding; in the cases where a phenyl group spacer exists, the aromatic ring in some cases actually enhances binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylgalactosamine↗
PubMed1989

Interaction of glyceraldehyde-3-phosphate dehydrogenase with AMP as studied by means of a spin-labeled analog.

The binding of a spin-labeled AMP analog to tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle is described. The spin label, perdeuterated and 15N-substituted 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, was attached to C-8 of AMP (C8-SL-AMP). Up to 8 equivalents of C8-SL-AMP bind per enzyme tetramer, i.e., 2 per monomer. Combining sites are the adenine subsite of the coenzyme-binding domain and the phosphate site. Glyceraldehyde 3-phosphate causes a conformational change in the enzyme that brings C8-SL-AMP molecules bound to adjacent R-axis-related subunits closer to one another by 0.2-0.3 nm and allows for spin-spin interaction between the nitroxide radicals. Similar, but less pronounced structural changes take place upon lowering the pH from 8 to 7. Addition of a single equivalent of NAD+ to a complex of the enzyme with 7.6 equivalents of C8-SL-AMP leads to the release of almost 4 C8-SL-AMP molecules. This supports our previous findings that binding of just one NAD+ molecule induces conformational changes in all four subunits.

Adenosine Monophosphate↗
PubMed1981

Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability.

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.

Animals↗
PubMed1994

A spin label that binds to myosin heads in muscle fibers with its principal axis parallel to the fiber axis.

We have used an indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione), designated InVSL, to study the orientation of myosin heads in bundles of chemically skinned rabbit psoas muscle fibers, with electron paramagnetic resonance (EPR) spectroscopy. After reversible preblocking with 5,5'-dithiobis(2-nitro-benzoic acid) (DTNB), we were able to attach most of the spin label covalently and rigidly to either Cys 707 (SH1) or Cys 697 (SH2) on myosin heads. EPR spectra of labeled fibers contained substantial contributions from both oriented and disordered populations of spin labels. Similar spectra were obtained from fibers decorated with InVSL-labeled myosin heads (subfragment 1), indicating that virtually all the spin labels in labeled fibers are on the myosin head. We specifically labeled SH2 with InVSL after reversible preblocking of the SH1 sites with 1-fluoro-2,4-dinitrobenzene (FDNB), resulting in a spectrum that indicated only disordered spin labels. Therefore, the oriented and disordered populations correspond to labels on SH1 and SH2, respectively. The spectrum of SH2-bound labels was subtracted to produce a spectrum corresponding to SH1-bound labels, which was used for further analysis. For this corrected spectrum, the angle between the fiber axis and the principal axis of the spin label was fitted well by a Gaussian distribution centered at theta o = 11 +/- 1 degree, with a full width at half-maximum of delta theta = 15 +/- 2 degrees. The unique orientation of InVSL, with its principal axis almost parallel to the fiber axis, makes it complementary to spin labels previously studied in this system. This label can provide unambiguous information about axial rotations of myosin heads, since any axial rotation of the head must be reflected in the same axial rotation of the principal axis of the probe, thus changing the hyperfine splitting. Therefore, InVSL-labeled fibers have ideal properties needed for further exploration myosin head orientation and rotational motion in muscle.

Animals↗
PubMed2002

Structural studies on membrane proteins using non-linear spin label EPR spectroscopy.

Non-linear electron spin resonance (EPR) techniques suitable for measuring proximity relationships in membranes are reviewed. These were developed during the past decade in order to measure changes sensitively in the spin-lattice relaxation time (T1) of nitroxyl spin labels covalently attached to membrane lipids or proteins. In combination with paramagnetic quenching agents and double spin-labelling, the methods were further developed for distance measurements. Selected examples are given to illustrate different methods, and types of data obtained for both integral and peripheral membrane proteins.

Electron Spin Resonance Spectroscopy↗
PubMed1982

Preparation and properties of spin-labeled lecithin-cholesterol liposomes.

Lecithin-cholesterol vesicles of various compositions containing membrane-bound spin-labeled cholestane can be prepared by appropriate choice of initial concentrations of components during sonication. Increasing incorporation of spin label increases incorporation of cholesterol and decreases incorporation of lecithin, with the result that liposomes with cholesterol-lecithin molar ratios larger than 2 can be obtained. Besides associating with cholesterol-lecithin complexes in the liposome, the spin label seems to associate with cholesterol. Changes of the paramagnetic resonance spectrum of the liposome-bound spin label due to changes in liposomal cholesterol and spin label mole fractions - assessed by three parameters - can be used in cell-liposome interaction studies.

Cholesterol↗
PubMed1999

Early perfusion after controlled cortical impact in rats: quantification by arterial spin-labeled MRI and the influence of spin-lattice relaxation time heterogeneity.

Early posttraumatic cerebral hypoperfusion is implicated in the evolution of secondary damage after experimental and clinical traumatic brain injury (TBI). This is the first report of cerebral blood flow (CBF) measurement by continuous arterial spin-labeled magnetic resonance imaging (MRI) early after TBI in rats using the controlled cortical impact (CCI) model. CCI reduced CBF globally at approximately 3 hr (versus normal), with 85% and 49% reductions in a contused cortical region and contralateral cortex, respectively. In contrast, a prior MRI study from this laboratory showed at 24 hr post trauma a focal CBF reduction restricted to the injury site. In vivo spin-lattice relaxation time (T(1obs)), which is used in CBF quantification, was spatially heterogeneous early after CCI, a time when edema is developing in injured brain tissue. At 4.7 T, T(1obs) values are increased 29% in the contusion (versus normal), consequently reducing CBF quantification to a similar degree. MRI should facilitate coupling posttraumatic CBF with long-term functional outcome. Magn Reson Med 42:673-681, 1999.

Animals↗
PubMed1985

Enhanced MRI of tumors utilizing a new nitroxyl spin label contrast agent.

Nitroxyl spin labels have been shown to be effective in vivo contrast agents for magnetic resonance imaging (MRI) of the central nervous system, myocardium, and urinary tract. A new pyrrolidine nitroxyl contrast agent (PCA) with better resistance to in vivo metabolic inactivation than previously tested agents was studied for its potential to enhance subcutaneous neoplasms in an animal model. Twenty-two contrast enhancement trials were performed on a total of 15 animals 4-6 weeks after implantation with human renal adenocarcinoma. Spin echo imaging was performed using a .35 T animal imager before and after intravenous administration of PCA in doses ranging from 0.5 to 3mM/kg. The intensity of tumor tissue in the images increased an average of 35% in animals receiving a dose of 3 mM/kg. The average enhancement with smaller doses was proportionately less. Tumor intensity reached a maximum within 15 min of injection. The average intensity difference between tumor and adjacent skeletal muscle more than doubled following administration of 3 mM/kg of PCA. Well-perfused tumor tissue was more intensely enhanced than adjacent poorly perfused and necrotic tissue.

Animals↗
PubMed2001

Distance mapping of protein-binding sites using spin-labeled oligosaccharide ligands.

The binding of a nitroxide spin-labeled analog of N-acetyllactosamine to galectin-3, a mammalian lectin of 26 kD size, is studied to map the binding sites of this small oligosaccharide on the protein surface. Perturbation of intensities of cross-peaks in the (15)N heteronuclear single quantum coherence (HSQC) spectrum of full-length galectin-3 owing to the bound spin label is used qualitatively to identify protein residues proximate to the binding site for N-acetyllactosamine. A protocol for converting intensity measurements to a more quantitative determination of distances between discrete protein amide protons and the bound spin label is then described. This protocol is discussed as part of a drug design strategy in which subsequent perturbation of chemical shifts of distance mapped amide cross-peaks can be used effectively to screen a library of compounds for other ligands that bind to the target protein at distances suitable for chemical linkage to the primary ligand. This approach is novel in that it bypasses the need for structure determination and resonance assignment of the target protein.

Amino Sugars↗
PubMed1978

Cardiolipin: a stereospecifically spin-labeled analogue and its specific enzymic hydrolysis.

The spin-labeled cardiolipin 1-(3-sn-phosphatidyl)-3-[1-acyl-2-(16-doxylstearoyl)glycero(3)phosphol]-sn-glycerol has been prepared. The stereoselective synthesis makes use of the monolysocardiolipin 1-(3-sn-phosphatidyl)-3-[1-acyl-2-lyso-sn-glycero(3)phospho]-sn-glycerol, available from the stereospecific hydrolysis of cardiolipin by phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) of Trimeresurus flavoviridis. The results of treatment of the spin-labeled cardiolipin with the cardiolipin-specific phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) (Hemophilus parainfluenzae) of known specificity and with phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) of Bacillus cereus are consistent with the assigned structure. The spin-labeled cardiolipin is further characterized and the unique features of this diastereomer are discussed in the context of the unusual stereochemistry of the natural phospholipid.

Cardiolipins↗
PubMed1982

The effects of glutaraldehyde and osmium tetroxide on the erythrocyte membrane. A spin label study.

A nitroxide spin label probe technique was applied to study the interaction between glutaraldehyde or osmium tetroxide (OsO4) amd the membranes of horse erythrocytes, ghosts and liposomes prepared from erythrocyte lipids. Two major conclusions have been established: (1) Reaction of the fixation reagents with the membrane is selective. OsO4 reacts predominantly with lipids and glutaraldehyde with membrane proteins. (2) The lipid-protein interactions change after pretreatment by OsO4 or glutaraldehyde.

Aldehydes↗
PubMed2002

Structure of spin-labeled methylmethanethiolsulfonate in solution and bound to TEM-1 beta-lactamase determined by electron nuclear double resonance spectroscopy.

Site-directed spin-labeling of proteins whereby the spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)methanethiolsulfonate (SLMTS) is reacted with the -SH groups of cysteinyl residues incorporated into a protein by mutagenesis has been successfully applied to investigate secondary structure and conformational transitions of proteins. In these studies, it is expected that the spin-label moiety adopts different conformations dependent on its local environment. To determine the conformation of SLMTS in solution reacted with L-cysteine (SLMTCys) and bound in the active site of the Glu240Cys mutant of TEM-1 beta-lactamase, we have synthesized SLMTS both of natural abundance isotope composition and in site-specifically deuterated forms for electron nuclear double resonance (ENDOR) studies. ENDOR-determined electron-proton distances from the unpaired electron of the nitroxyl group of the spin-label to the methylene and methyl protons of SLMTS showed three conformations of the oxypyrrolinyl ring with respect to rotation around the S-S bond dependent on the solvent dielectric constant. For SLMTCys, two conformations of the molecule were compatible with the ENDOR-determined electron-nucleus distances to the side-chain methylene protons and to H(alpha) and H(beta1,2) of cysteine. To determine SLMTS conformation reacted with the Glu240Cys mutant of TEM-1 beta-lactamase, enzyme was overexpressed in both ordinary and perdeuterated minimal medium. Resonance features of H(alpha) and H(beta1,2) of the Cys240 residue of the mutant and of the side-chain methylene protons within the spin-label moiety yielded electron-proton distances that sterically accommodated the two conformations of free SLMTCys in solution.

Binding Sites↗
PubMed1996

[Conformation properties of heme-containing proteins using spin labels].

The review briefly summarizes the results of spin-label studies of conformational transitions in monomeric globins (myoglobin, leghemoglobin, erythrocruorin) as well as in another heme-containing protein, cytochrome c, induced by heme ligands and pH. In contrast with integral methods of investigation, for proteins with a known spatial structure the use of the spin-labelling technique makes it possible to obtain information on the conformational behaviour of the isolated parts of the protein structure which, alongside with the results obtained by other physico-chemical methods, allows a conclusion on the nature of conformational movements in the protein during its functioning. The experimental results fit well into the model which represents a Mb-like structure as composed of three independent rigid helical fragments: AE(ABCDE), F and GH, whose reciprocal arrangement is controlled by N- and C-terminal salt bridges. Synchronous displacement of the above fragments relative to one another, which is due either to the ligand attachment and structural changes in the heme complex or to disturbances in ionic interactions at the N- and/or C-end(s) of the structure under effect of pH or allosteric effectors, might serve as a structural basis for homo- and heterotropic regulation in hemoglobin. Similarly, the spin label method allows for tracing and obtaining important information about local conformational events in cytochrome c which in the native protein are associated with a change in pH in the range of 5-13 and are accompanied by the substitution of the heme sixth ligand, Met-80, by Lys-79 (pH 9.3), and then by Tyr-67 (pK 11.1). Local changes in the conformation and dynamic properties of native cytochrome c provide, if necessary, global changes in its structure upon alkaline denaturation. It was found that substitution of the heme protein ligand, Met-80, by the external ligand, cyanide, markedly alters the dynamic properties of the polypeptide chain segment 67-75 adjacent to Met-80.

Hemeproteins↗
PubMed2000

Assessment of cerebral blood flow in Alzheimer's disease by spin-labeled magnetic resonance imaging.

To evaluate the utility of arterial spin-labeled blood flow magnetic resonance imaging for the detection of cerebral blood flow abnormalities in Alzheimer's disease, arterial spin-labeled blood flow images in 16 contiguous 5-mm axial sections were acquired in 18 patients diagnosed with probable Alzheimer's disease and 11 age-matched controls. Blood flow images from all subjects were transformed to a standard anatomical space for voxel-by-voxel statistical analysis. High quality blood flow images were obtained from all but 1 subject. Statistical analysis demonstrated significant flow decreases relative to control subjects in temporal, parietal, frontal, and posterior cingulate cortices. Increased severity of disease, as measured by Mini-Mental State Examination, correlated with posterior parietal and posterior cingulate decreases but not temporal decreases. Arterial spin-labeled magnetic resonance imaging was found to be an effective tool for characterizing flow decreases accompanying Alzheimer's disease. The absence of ionizing radiation or injection and the ability to obtain high quality anatomical images within the same scanning session make arterial spin labeling an attractive technique for the study of Alzheimer's disease, for the evaluation of pharmacological therapies, and, possibly, for early diagnosis.

Aged↗
PubMed1987

Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids.

This report describes a method suitable for determining the depth of a wide variety of fluorescent molecules embedded in membranes. The method involves determination of the parallax in the apparent location of fluorophores detected when quenching by phospholipids spin-labeled at two different depths is compared. By use of straightforward algebraic expressions, the method allows calculation of depth in angstroms. Furthermore, the analysis can be extended to quenching by energy-transfer acceptors or brominated probes under appropriate conditions. Application of the method to quenching of 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled lipids by spin-labeled lipids located at three different depths is demonstrated in model membranes. It is shown that the calculated depths of the NBD groups are self-consistent to the extent that they are the same no matter which two spin-labels have been used in a particular experiment. In addition, the calculated depth is independent of spin-label concentration in the membrane within +/- 1 A, ruling out major effects due to spin-label perturbation. The quenching experiments show that the location of the NBD group in head-group-labeled phosphatidylethanolamine is at the polar/hydrocarbon interface and that of an NBD label on the "tail" of cholesterol is deeply buried, as expected. Unexpectedly, NBD labels placed at the end of fatty acyl chains of phosphatidylcholines are also near the polar/hydrocarbon interface. Presumably, the polarity of the NBD group results in "looping" back to the surface of the NBD groups attached to flexible acyl chains.

4-Chloro-7-nitrobenzofurazan↗
PubMed1988

Characterization of protein spin labeling by maleimide: evidence for nitroxide reduction.

A quantitative determination of maleimide spin label (MAL) binding in oxi and met hemoglobin (Hb) and bovine serum albumin are investigated using double integration to the ESR signal. This determination permitted the observation that a considerable fraction of MAL is reduced, losing its paramagnetism. Experiments using the same spin label with myoglobin and Hb with blocked-SH groups, where reduction was not observed, indicate the involvement of SH groups in the process. The 4-hydroxy-2,2,6,6-tetramethylpiperidino-1-oxyl spin label (which is not able to bind in the SH group) is reduced too, but the dependence on the molar ratio is different in comparison with the MAL case. In both cases the reduction percentage depends on the molar ratio spin label to protein and to the protein concentration. In order to obtain the total SH groups labeled (two in the Hb case) it is necessary to use an excessive amount of label (around 18:1) in the 0.5 mM Hb concentration.

Cyclic N-Oxides↗
PubMed1994

[The protective effects of metallothionein on membrane proteins against endothelin--electron paramagnetic resonance spin labelling study].

Using EPR spin labelling 5 NS, 16 NS (for fatty acids) and MSL (for proteins), the changes in lipid fluidity and protein conformation in the ghost erythrocyte membrane of rat treated by endothelin have been studied. The results showed that the lipid fluidity in both of the polar region and the hydrophobic core in the membranes was not altered by endothelin at the concentration of 1 x 10(-9), 1 x 10(-8) and 1 x 10(-7) mol/L, but the ratio of S/W of membrane proteins was significantly changed by endothelin at 5 x 10(-9) and 1 x 10(-7) mol/L. This means that endothelin at the dosages used did not affect the membrane lipids but did alter protein conformation. Metallothionein (1 x 10(-5) mol/L) could attenuate this change and protect membrane proteins from endothelin. However, there is no effect of metallothionein on membrane proteins.

Animals↗