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PubMed2005

ESR spin-label study of poly(styrene-co-methacrylic acid)/poly(epsilon-caprolactone) semi-interpenetrating polymer networks with controlled hydrogen-bond interactions.

The morphology and miscibility of semi-interpenetrating polymer networks (semi-IPN) prepared with poly(styrene-co-methacrylic acid) [P(S-co-MAA)] of different carboxylic acid contents and poly(epsilon-caprolactone) (PCL) have been studied by ESR spin-label method. The ESR spectra of spin-labeled PCL showed one motional component at any specific temperature. It indicated that the spin-labeled molecules were located in one type of environment. The coexistence of two motional components in the ESR spectra of all semi-IPN samples was observed over a certain temperature range. This phenomenon suggested that the semi-IPNs were not compatible systems; they contained two microphases, a PCL-rich microdomain and a P(S-co-MAA)-rich microdomain. The miscibility could be improved by increasing the carboxylic acid content, which could enhance the hydrogen-bonding interactions between the ester groups of PCL and carboxylic acid groups in P(S-co-MAA). It was also found that the intracomponent cross-linking of the semi-IPNs was not in favor of the miscibility. The microphase separation occurred in all semi-IPNs, even in the samples having strong hydrogen-bonding interactions. With increasing cross-linking density, the microphase separation became more remarkable.

Electron Spin Resonance Spectroscopy↗
PubMed1975

Molecular interactions of adenosine triphosphatase with the mitochondrial membrane as revealed by a spin label study.

Mitochondrial ATPase complex has been spin-labeled in the membrane using the inhibitor N-(2,2,6,6-tetramethylpeperidyl-1-OXYL)-N(cyclohexyl)carbodiimide (nccd). the amount of NCCD bound to mitochondrial fragments is 0.5 nmol/mg and cannot be dialyzed or extracted with ether, chloroform, or methanol. The electron paramagnetic resonance spectrum of NCCD bound to fragments is pH-sensitive, a greater label immobilization occurring at pH values lower or higher than 7. Ether extraction removes the ATPase inhibition by NCCD without detaching the label. This effect appears to be the consequence of the dislocation of some components of the ATPase complex. Removal of F1 natural inhibitor or of F1 does not affect the spectrum of NCCD bound to fragments, while the removal of oligomycin sensitivity-conferring protein produces an increase in the extreme splitting. Oligomycin sensitivity-conferring protein may thus interact with the NCCD binding component of the membrane. The isolation of the NCCD-binding proteolipid results in a large increase in the mobility of the label, but addition of dipalmitoyllecithin decreases the mobility of the label to the original level. Phospholipids are thus necessary to keep the NCCD-binding proteolipid in the native conformation.

Adenosine Triphosphatases↗
PubMed1986

Association of gangliosides with the lymphocyte plasma membrane studied using radiolabels and spin labels.

Gangliosides are known to act as potent suppressors of lectin-stimulated lymphocyte activation when added to the culture medium. Since this effect may be mediated via ganglioside association with (or insertion into) the plasma membrane, we have used 3H- and spin-labelled derivatives of mixed gangliosides to probe the nature of this interaction. Gangliosides bind rapidly to the lymphocyte membrane and show no preference for association with either inside-out or right-side-out membrane vesicles. Around 20% of the bound gangliosides can be removed by repetitive washing, and a further 22-28% by treatment with pronase for 1 h, suggesting that this fraction is tightly bound to membrane proteins at the cell surface. The ESR spectrum of membrane-bound gangliosides did not resemble the spin-exchanged spectrum of micellar spin-labelled gangliosides in aqueous solution, but was similar to that seen for 5 mol% ganglioside spin label in liposomes of egg phosphatidylcholine. This suggests that the bulk of the membrane-bound gangliosides are inserted and molecular dispersed in the lymphocyte membrane. Binding of wheat-germ agglutinin to lymphocyte-associated gangliosides results in specific immobilization of the carbohydrate headgroup, while concanavalin A and other lectins have little or no effect on oligosaccharide mobility. Membrane-inserted gangliosides show a response to lectin binding which is qualitatively different from that seen for gangliosides in bilayers of phosphatidylcholine.

Animals↗
PubMed1982

Synthesis of charged amphipathic nitroxide lipid spin labels and an example of their application in membrane studies.

The synthesis of a series of amphipathic nitroxide lipid spin labels is reported. Thus, 12-proxylhexadecanol has been converted into the versatile fatty acid spin label 14-proxylstearic acid. This substance was used to prepare 14-proxylstearyltrimethylammonium methanesulfonate, a positively charged label, and 14-proxylstearylmethyl phosphate sodium salt, a negatively charged label. Also prepared in the doxyl series were quaternary ammonium salts derived from 16-doxyl- and 7-doxylstearic acid. The positively charged and negatively charged proxyl labels were used in a preliminary experiment to investigate the role of charge in their interaction with reconstituted cytochrome oxidase. The average binding affinity of the negatively charged label is approximately 2-fold higher than that of the positively charged label at pH 7.4. At pH 5.5 the average relative affinity for negatively charged label is about 3.5-fold higher than that of positively charged label, suggesting that the ionizable group(s) on the protein can interact with the lipid headgroup.

Cyclic N-Oxides↗
PubMedDate not supplied

[Spin-label progesterone binding to serum albumin].

The binding of spin label progesterone to bovine serum albumin was studied by the spin-probe technique. The binding capacity of protein was established. It was shown that protein formed a rigid complex with steroid, the correlation time of this complex being 50 ns. In the complex the radical part of the steroidal molecule has a hydrophobic environment.

Electron Spin Resonance Spectroscopy↗
PubMed2006

Site-directed parallel spin-labeling and paramagnetic relaxation enhancement in structure determination of membrane proteins by solution NMR spectroscopy.

A major challenge for the structure determination of integral membrane proteins by solution NMR spectroscopy is the limited number of NOE restraints in these systems stemming from extensive deuteration. Paramagnetic relaxation enhancement (PRE) by means of nitroxide spin-labels can provide valuable long-range distance information but, in practice, has limits in its application to membrane proteins because spin-labels are often incompletely reduced in highly apolar environments. Using the integral membrane protein OmpA as a model system, we introduce a method of parallel spin-labeling with paramagnetic and diamagnetic labels and show that distances in the range 15-24 Angstroms can be readily determined. The protein was labeled at 11 water-exposed and lipid-covered sites, and 320 PRE distance restraints were measured. The addition of these restraints resulted in significant improvement of the calculated backbone structure of OmpA. Structures of reasonable quality can even be calculated with PRE distance restraints only, i.e., in the absence of NOE distance restraints.

Bacterial Outer Membrane Proteins↗
PubMed2006

Orientation of TOAC amino-acid spin labels in alpha-helices and beta-strands.

The orientation of alpha-helices or beta-strands, e.g., in membranes, can be determined from EPR order parameters of (2,2,6,6-tetramethyl-piperidine-1-oxy-4-amino-4-carboxylic acid) TOAC amino-acid spin labels incorporated in the polypeptide backbone. This requires knowledge of the inclination of the nitroxide axes, relative to the alpha-helix or beta-strand axis. Crystal structures of TOAC-containing peptides are used to derive the spin-label orientation relative to refined alpha-poly-l-alanine and beta-poly-l-alanine structures. The spin-label z-axes of the two mirror-image TOAC twist-boat conformers are inclined at 13+/-2 degrees and 65+/-3 degrees , respectively, to the alpha-helix axis, or at 25+/-3 degrees and 32+/-3 degrees to the beta-strand axis.

Alanine↗
PubMed1997

Membrane location of spin-labeled M13 major coat protein mutants determined by paramagnetic relaxation agents.

Mutants of the M13 bacteriophage major coat protein containing single cysteine replacements (A25C, V31C, T36C, G38C, T46C, and A49C) in the hydrophobic and C-terminal domains were purified from viable phage. These were used for site-directed spin-labeling to determine the location and assembly of the major coat protein incorporated in bilayer membranes of dioleoylphosphatidylcholine. The membrane location of the spin-labeled cysteine residues was studied with molecular oxygen and Ni2+ ions as paramagnetic relaxation agents preferentially confined to the hydrophobic and aqueous regions, respectively, by using progressive-saturation electron spin resonance (ESR) spectroscopy. The section of the protein around Thr36 is situated at the center of the membrane. Residue Thr46 is placed at the membrane surface in the phospholipid head group region with a short C-terminal section, including Ala49, extending into the aqueous phase. Residue Ala25 is then positioned consistently in the head group region of the apposing lipid monolayer leaflet. These positional assignments are consistent with the observed mobilities of the spin-labeled groups. The outer hyperfine splittings in the ESR spectra decrease from the N-terminal to the C-terminal of the hydrophobic section (residues 25-46), and then drop abruptly in the aqueous phase (residue 49). Additionally, the strong immobilization and low oxygen accessibility of residue 25 are attributed to steric restriction at the hinge region between the transmembrane and N-terminal amphipathic helices. Sequence-specific modulations of the ESR parameters are also observed. Relatively low oxygen accessibilities in the hydrophobic region suggest intermolecular associations of the transmembrane helices, in agreement with saturation transfer ESR studies of the overall protein mobility. Relaxation enhancements additionally reveal a Ni2+ binding site in the N-terminal domain that is consistent with a surface orientation of the amphipathic helix.

Binding Sites↗
PubMed1986

Spin label study of local anesthetic-lipid membrane interactions. Phase separation of the uncharged form and bilayer micellization by the charged form of tetracaine.

The interaction between tetracaine and egg phosphatidylcholine (egg PC) multibilayers was examined. ESR spectra of an ester spin label indicate that at low uncharged anesthetic: lipid ratios, membrane organization decreases. At higher ratios, saturation and phase separation occur, as suggested by a second spectral component which appears when the water solubility of tetracaine is reached. However, experiments with the drug in the absence and in the presence of membranes, making use of a phospholipid spin label, suggest that the new phase does not consist of solid tetracaine alone. Location of the new phase in the membrane would require a change in partition coefficient, while its location outside would imply a mechanism whereby the anesthetic would come off the membrane as an aggregate containing spin probe and phospholipid. Charged tetracaine forms micelles which disrupt-unilamellar egg PC vesicles (Fernandez, M.S. (1981) Biochim. Biophys. Acta 646, 27-30). Micellar tetracaine added to bilayers containing a PC spin probe changes the spectrum from one typical of a bilayer into one typical of micelles, indicating the formation of a tetracaine-egg PC mixed micelle. The effect is reversible upon dilution to concentrations below the critical micelle concentration of tetracaine. When membranes are prepared in the presence of a water-soluble spin label, TEMPOcholine, ascorbate destroys the signal of untrapped label; when mixed phospholipid-tetracaine are formed by addition of micellar tetracaine, this leads to a complete loss of the ESR signal. High drug concentrations are often used for anesthesia and could be related to morphological nerve damage caused by large doses of anesthetics.

Anesthetics, Local↗
PubMed2003

Structure of the substrate binding pocket of the multidrug transporter EmrE: site-directed spin labeling of transmembrane segment 1.

Site-directed spin labeling (SDSL) was used to explore the structural framework responsible for the obligatory drug-proton exchange in the Escherichia coli multidrug transporter, EmrE. For this purpose, a nitroxide scan was carried out along a stretch of 26 residues that include transmembrane segment 1 (TMS1). This segment has been implicated in the catalytic mechanism of EmrE due to the presence of the highly conserved glutamate 14, a residue absolutely required for ligand binding. Sequence-specific variation in the accessibilities of the introduced nitroxides to molecular oxygen reveals a transmembrane helical conformation along TMS1. One face of the helix is in contact with the hydrocarbon interior of the detergent micelle while the other face appears to be solvated by an aqueous environment, resulting in significant exposure of the nitroxides along this face to NiEDDA. TMS1 from two different subunits are in close proximity near a 2-fold axis of symmetry as revealed by the analysis of spin-spin interactions at sites 14 and 18. The limited extent of spin-spin interactions is consistent with a scissor-like packing of the two TMS1. This results in a V-shaped chamber which is in contact with the aqueous phase near the N-terminus. The spatial organization of TMS1, particularly the close proximity of E14, is consistent with a proposed mechanistic model of EmrE [Yerushalmi, H., and Schuldiner, S. (2000) Biochemistry 39, 14711-14719] where substrate extrusion is coupled to proton influx through electrostatic interactions and shifts of the glutamate 14 pK(a) during the cycle.

Antiporters↗
PubMed2004

Myocardial perfusion measurements by spin-labeling under different vasodynamic states.

In this study absolute myocardial perfusion was determined using a spin-labeling magnetic resonance imaging (MRI) technique at 2 Tesla. The technique was applied to 16 healthy volunteers at resting conditions, adenosine-induced stress, and oxygen breathing. Overall myocardial quantitative perfusion was determined as 2.3 +/- 0.8 mL/g/min (rest), 4.2 +/- 1.0 mL/g/min (adenosine), and 1.6 +/- 0.6 mL/g/min (oxygen), respectively. T1 of left ventricular blood pool decreased from 1709 +/- 101 ms (rest) to 1423 +/- 61 ms (oxygen), whereas T1 of right ventricular blood did not change significantly (1586 +/- 126 ms and 1558 +/- 150 ms). In conclusion, the presented technique for quantification of myocardial perfusion is an alternative to contrast agent-based methods. The spin labeling method is noninvasive and easily repeatable and it could therefore become an important tool to study changes in myocardial perfusion under different vasodynamic states.

Adenosine↗
PubMed2005

Experience in implementing continuous arterial spin labeling on a commercial MR scanner.

Continuous arterial spin labeling (CASL) is a technique for performing quantitative perfusion measurements without the need for exogenous contrast agent administration. This technique has seen limited use in the clinic due to problems of poor sensitivity and the potential for artifacts. In addition, CASL requires the application of long-duration radiofrequency pulses and the acquisition of a large number of images, which can cause difficulties when implemented on commercial MR scanners. This work details our experience in implementing CASL on a commercial MR scanner for the measurement of cerebral blood flow, including pitfalls regarding hardware, radiofrequency energy deposition, and practical application in human subjects. Results of studies to determine the optimal acquisition procedures are also presented.

Adult↗
PubMed1997

Molecular distances from dipolar coupled spin-labels: the global analysis of multifrequency continuous wave electron paramagnetic resonance data.

For immobilized nitroxide spin-labels with a well-defined interprobe geometry, resolved dipolar splittings can be observed in continuous wave electron paramagnetic resonance (CW-EPR) spectra for interelectron distances as large as 30 A using perdeuterated probes. In this work, algorithms are developed for calculating CW-EPR spectra of immobilized, dipolar coupled nitroxides, and then used to define the limits of sensitivity to the interelectron distance as a function of geometry and microwave frequency. Secondly, the CW-EPR spectra of N epsilon-spin-labeled coenzyme NAD+ bound to microcrystalline, tetrameric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been collected at 9.8, 34, and 94 GHz. These data have been analyzed, using a combination of simulated annealing and global analysis, to obtain a unique fit to the data. The values of the intermitroxide distance and the five angles defining the relative orientation of the two nitroxides are in reasonable agreement with a molecular model built from the known crystal structure. Finally, the effect of rigid body isotropic rotational diffusion on the CW-EPR spectra of dipolar coupled nitroxides has been investigated using an algorithm based on Brownian dynamics trajectories. These calculations demonstrate the sensitivity of CW-EPR spectra to dipolar coupling in the presence of rigid body rotational diffusion.

Algorithms↗
PubMedDate not supplied

[Interaction of spin-labeled methacyne analog with butyrylcholinesterase].

Interaction between spin-labeled methacyne (I) and butyrylcholinesterase (BChE) was studied by ESR and enzyme kinetic methods. The compound (I) was shown to be a competitive reversible inhibitor, the value of Ki appeared to be 1.3 X 10(-5) M. Insertion of nitroxyl fragment in the methacyne molecule results in a two-fold increase of its inhibitory activity. The ESR spectrum of the enzyme-inhibitor complex was registered. This complex dissociates under the action of eserine, tetramethylammonium and hexamethonium. Scatchard plot reveals two different types of binding sites with Kdiss values 1.5 X 10(-5) M and 2.6 X 10(-4) M. One type of binding sites is identified as the enzyme active centre. The restricted motion of (I) in complex with BChE proves the assumption that the enzyme active centre is located in the split of macromolecule surface.

Binding Sites↗
PubMedDate not supplied

[Stabilization of albumin by CaCl2- and MgCl2-regulated interaction of macromolecules: a study by the spin label method].

Effective thermodynamic activation parameters of spin-label transitions between microsurroundings in water-protein matrix of modified molecules of serum albumin are determined in 0.01 M acetate buffer, pH 5.6, in CaCl2 and MgCl2 concentration range from 10(-3) to 5 M and protein concentration 90-110 mg/ml. The "melting" of water-protein matrix being accompanied by disjoining pressure in water-protein matrix and native protein structure stability alterations is registered in the salt concentration range 0.1-0.3 M. The phenomena revealed are interpreted as phase transition from solution of protein clusters at the low salt concentrations to the "salting out" form of protein solution at high salt concentrations or to the solution in which interactions between proteins are stabilized by repulsive structural forces of residual hydration shells of the bound cations. This transition realizes through the intermediate (critical type) state characterized by weak protein-protein interactions. The flexibility of water-protein matrix and its permeability for the salt ions and water molecules is more pronounced in this region of moderate (approximately equal to 0.3 M) CaCl2 and MgCl2 concentrations.

Calcium Chloride↗
PubMed1993

Transport of spin-labeled tetracycline across model and biological membranes.

Electron spin resonance spectra of spin labeled tetracycline (TC-SL) do not show any recognizable partitioning into a lipid bilayer or bulk hydrocarbon solvent, paraffin oil. TC-SL, however, penetrates through the model and biological membranes. It is shown that the rate of permeation depends on membrane composition and increases with temperature. In fluid phase, the rate is greater for saturated dimyristoylphosphatidylcholine than for unsaturated egg yolk phosphatidylcholine membranes. Cholesterol significantly decreases the rate, 30 mol% cholesterol decreases the rate of TC-SL permeation across egg yolk phosphatidylcholine bilayer 8 times at 37 degrees C. The rate of permeation of TC-SL across model membranes is much smaller than the rate for TEMPONE--a compound which slightly partitions into lipid bilayer, and much greater than the rate for TEMPO--choline-a positively charged compound. After addition to the suspension of Ehrlich's ascites tumor cells, the TC-SL is reduced to a non-paramagnetic form. Reduction rate is independent of oxygen concentrations, which led us to suggest that the permeation of TC-SL across the cell plasma membrane is the limiting step of reduction reaction.

Animals↗
PubMed1990

Fluidity of rat liver Golgi membranes in streptozotocin diabetes. A spin label study.

The mobility of 5-doxylstearic acid spin label (5-SASL) in the intact rat liver Golgi membranes of streptozotocin diabetes was studied as a function of free blood sugar level and temperature. During development of diabetes, indicated by the increase of the free blood sugar level, the membrane fluidity measured in the physiological temperature range (1) does not change in comparison with control in light diabetes, (2) decreases significantly in advanced diabetes and (3) again increases to the control level in heavy diabetes (the free blood sugar levels being 200-250 mg/100 ml, 250-350 mg/100 ml and greater than 350 mg/100 ml, respectively). The development of streptozotocin diabetes is accompanied by significant changes in lipid composition of liver Golgi membranes as also shown in our previous observations. The measurements of motion of 5-SASL in Golgi membranes as well as in vesicles, made from commercially available lipids of composition close to the liver Golgi membranes, show that a decrease of cholesterol contents is the main factor which induces the increase membrane fluidity. We suggest that in the heavy diabetes the hemostatic regulation in the lipid composition leads to minimization of alterations in membrane fluidity to obtain comparatively normal activity of certain membrane enzymes.

Animals↗
PubMed1990

Fluidity of the phospholipid bilayer of the endometrium at the time of implantation of the blastocyst--a spin label study.

The endometrial phospholipid bilayer is shown to be in a highly fluid and polar state at the time of implantation in Mus musculus as evidenced from a spin label study using the stearic acid spin label, 5-doxyl stearate. The positive correlation observed between the superoxide anion radical levels and the fluidity and polarity state of the endometrial cells points towards the possibility of the mediation of this condition of unsaturation of fatty acids to be an act of the superoxide anion radical.

Animals↗