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PubMed1988

Bis(sulfo-N-succinimidyl) [15N,2H16]doxyl-2-spiro-4'-pimelate, a stable isotope-substituted, membrane-impermeant bifunctional spin label for studies of the dynamics of membrane proteins: application to the anion-exchange channel in intact human erythrocytes.

We have synthesized and characterized an isotopically substituted homologue of the membrane-impermeant bifunctional spin label bis(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate (BSSDP) [Beth et al. (1986) Biochemistry 25, 3824-3832] in which the nitroxide N is substituted with 15N and all of the protons in the doxylpimelate moiety are replaced by deuterons ([15N,2H16]BSSDP). Like its normal isotope homologue, [15N,2H16]BSSDP reacts with the anion-exchange channel in intact human erythrocytes at a site that spans the single extracytoplasmic chymotryptic cleavage site and that overlaps the stilbenedisulfonate site. The narrower line widths in the EPR spectrum of [15N,2H16]BSDP-labeled anion channels allow calculation of a minimum separation of 16 A between spin labels bound at the functionally important stilbenedisulfonate sites on adjacent subunits of an anion channel dimer. The 15N and 2H isotopic substitutions also provide substantial improvement in signal to noise of motionally sensitive regions of the ST-EPR spectrum of [15N,2H16]BSSDP-labeled anion channels in intact erythrocytes. [15N,2H16]BSSDP-labeled anion channels in intact erythrocytes were cross-linked to covalent dimers in the extracytoplasmic domain with the membrane-impermeant cross-linking reagent bis(sulfo-N-succinimidyl) suberate [Staros (1982) Biochemistry 21, 3950-3955], and the saturation-transfer EPR spectrum of these cells was compared with that of cells treated with [15N,2H16]BSSDP but not subsequently cross-linked. The spectra were essentially identical, supporting the hypothesis that anion channel subunits form stable dimers in the membranes of intact erythrocytes.

Anions↗

PubMed1997

Spin-label studies on the anchoring and lipid-protein interactions of avidin with N-biotinylphosphatidylethanolamines in lipid bilayer membranes.

The specific binding of hen egg white avidin to phosphatidylcholine lipid membranes containing spin-labeled N-biotinylphosphatidylethanolamines (biotin-PESLs) was investigated by using ESR spectroscopy. Spin-labeled biotin-PEs were prepared with the nitroxide group at position C-5, C-8, C-10, C-12, or C-14 of the sn-2 chain and were incorporated at 1 mol % in lipid bilayer membranes of dimyristoylphosphatidylcholine. Binding of avidin produced a strong and selective restriction of the biotin-PE lipid mobility at all positions of chain labeling, as shown by the ESR spectra recorded in the fluid lipid phase. The spectral components of the fraction of the biotin-PESLs that were not complexed by avidin indicated that the mobility of the bulk membrane lipids was unperturbed by binding avidin, as demonstrated by difference spectroscopy. Comparison of the positional profiles and temperature dependences of the outer hyperfine splittings from the biotin-PESLs suggests that the C-12 and C-14 positions of the avidin-bound biotin-PEs are in register with the C-5 and C-7/C-6 positions, respectively, of the chains of the bulk membrane lipids. This indicates that the biotin-PEs are partially withdrawn from the membrane, with a vertical displacement of ca. 7-8 A, on complexation with avidin. In addition, the specific lipid-protein interaction with avidin results in a selective reduction in the rates of lipid chain motion, as shown by the increased ESR line widths. These data define the way in which avidin is anchored to lipid membranes containing biotin-PEs.

Avidin↗

PubMed1996

Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics.

Thirty single cysteine substitution mutants of T4 lysozyme have been prepared and spin-labeled with a sulfhydryl-specific nitroxide reagent in order to systematically investigate the relationship between nitroxide side-chain mobility and protein structure. The perturbation caused by replacement of a native residue with a nitroxide amino acid was assessed from the resulting changes in biological activity, circular dichroism, and free energy of folding. The nitroxide produced context-dependent changes in stability and activity similar to those observed for substitution with natural amino acids at the same site but had little effect on the circular dichroism spectra. At solvent-exposed sites, the structural perturbation appears to be small at the level of the backbone fold. Nitroxide side-chain mobility faithfully reflects the protein tertiary fold at all sites investigated. The primary determinants of nitroxide side-chain mobility are tertiary interactions and backbone dynamics. Tertiary interactions constrain the side-chain mobility to an extent closely correlated with the degree of interaction. At interhelical loop sites, the side chains have a high mobility, consistent with high crystallographic thermal factors. On the exposed surfaces of alpha-helices, the side-chain mobility is not restricted by interactions with nearest neighbor side chains but appears to be determined by backbone dynamics. An unexpected result is a striking difference between the mobility of residues near the C- and N-termini of helices. These results provide the foundation for another dimension of information in site-directed spin-labeling experiments that can be interpreted in terms of the protein tertiary fold, its equilibrium dynamics and time-dependent conformational changes.

Bacteriophage T4↗

PubMed1984

Binding of a spin label analogue of compound 48/80 to rat peritoneal mast cells: correlation of binding properties with surface topography.

A spin label analogue of compound 48/80 has been synthesized and its binding to purified rat peritoneal mast cells has been studied by electron spin resonance spectroscopy. The spin label analogue (SL-48/80) had almost identical biological activity to unlabeled compound 48/80. SL-48/80 was used to estimate the number of binding sites per cell on normal mast cells (7.25 X 10(10)), on mast cells deactivated by sodium azide and 2-deoxyglucose or by heating to 46 degrees for 30 min (1 X 10(10)) and cells from animals actively-sensitized to ovalbumin (5.2 X 10(10)). SL-48/80 was also shown to bind to isolated mast cell granules. Differences in the binding properties of mast cells after the different treatments are related to their surface topography as seen by scanning electron microscopy, and the contribution of the granules to the number of binding sites is discussed.

Animals↗

PubMed1976

Interactions of the cholesterol side-chain with egg lecithin. A spin label study.

The effect in egg lecithin liposomes of cholesterol and cholesterol analogues with side-chains of reduced length on the order parameters of two steroid spin labels has been studied. Analogues with side-chains shorter than cholesterol by more than three carbon cause significantly less ordering than cholesterol. Liposomes containing a cholesterol analogue in which the side-chain is absent cause very little increase in the ordering of a new sterol spin label in which the nitroxide is incorporated into the side-chain. The results suggest that the sterol side-chain exerts a great influence on membrane rigidity within its immediate environment.

Binding Sites↗

PubMed2006

Spin-labeled alkylphospholipids in a dipalmitoylphosphatidylcholine bilayer: molecular dynamics simulations.

Molecular dynamics simulation has been performed to investigate the structural properties of perifosine and its synthetic spin-labeled alkylphospholipid analogues. The conformations adopted by these compounds in water and in a dipalmitoylphosphatidylcholine bilayer as a function of the presence and position of the N-oxyl-4',4'-dimethyloxazolidine ring (doxyl group) have been investigated by all-atom molecular dynamics. No predominant conformation was observed in water, but the molecules adopt specific orientations and conformations in the lipid bilayer. As is expected, alkyl chains tend to insert into the hydrophobic core, while charged groups stay at the lipid-water interface. A doxyl group in the middle of the alkyl chain moves up to the interface region, thus preventing adoption of the extended conformation. Compounds with a doxyl group close to the polar head group adopt conformations similar to that of unlabeled perifosine within the first nanoseconds of simulation. When the doxyl group is at the end of alkyl chain, the spin-labeled molecule needs more time to reach equilibrium. These results indicate a considerable effect of the doxyl position within the alkyl chain on its localization in the lipid bilayer and can be extended further to other similar spin probes used in the electron paramagnetic resonance spectroscopy of biological membranes.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1988

Hyposmolar entrapping of a spin label into aged red cells.

The aging of human erythrocytes stored in vitro at 4 degrees C was studied by the entrapping method. Erythrocytes were subjected to a sudden hyposmolar stress by suspension in solutions of varying osmolarity in the presence of the spin label tempocholine. The curves obtained when the amount of the spin label entrapped in the ghosts after resealing was plotted against the osmolarity of the buffer solutions exhibited an entrapping which increased with time of the in vitro storage of the erythrocytes (40-60 and 80-100 days). Rejuvenation of the aged erythrocytes by addition of glucose-adenine restored the entrapping curves to shapes nearly similar to those obtained with erythrocytes collected 30-35 days earlier.

Cells, Cultured↗

PubMedDate not supplied

Comparison of ionic and non-ionic nitroxide spin labels for urographic enhancement in magnetic resonance imaging.

Two nitroxide spin labels (NSL) were compared for in vitro relaxivity and in normal rats for efficiency of urographic enhancement. One of the NSL, PCA, a pyrrolidinyl agent, was ionic and one, NAT, was a non-ionic pyrrolidinyl NSL with multiple hydroxyl substituents for water solubility. Using both NSLs the renal medulla and papilla were noted to show greater contrast enhancement than the cortex, with a maximum enhancing effect between 5 and 15 minutes. Using doses of 1.0 and 2.5 mmol/kg, more than 100 per cent increases in spin echo intensities above the baseline were observed. The lowest tested dose of 0.1 mmol/kg showed an easily detectable enhancing effect for NAT. The good contrast enhancing properties of NAT, considered together with its better acute tolerance, justifies further investigation of this non-ionic compound.

Animals↗

PubMed1995

A quantitative analysis of alternated line scanning in k space and its application in MRI of regional tissue perfusion by arterial spin labeling.

A quantitative analysis of the alternated-line-scanning method is performed for k-space data acquisition in MRI. Application of the method in MRI of regional tissue perfusion by arterial spin labeling is demonstrated. It is also shown that, by using the alternated-line-scanning method, the degree of arterial spin labeling alpha can be determined in a single scan.

Animals↗

PubMed2000

Quantitation of renal perfusion using arterial spin labeling with FAIR-UFLARE.

Quantitative perfusion imaging of human kidneys was performed using arterial spin labeling MRI with a fast spin echo readout-sequence. Perfusion maps of centrally located single slices were obtained in axial and coronal orientations. In ten healthy volunteers, the mean value of perfusion was 213+/-55 mL/(100g min) with a range from 140 to 319 mL/(100g min). These results are in accordance with literature data, considering the fact that FAIR only measures the perfusion component normal to the imaging plane. Intra-individual reproducibility errors of +/-11% were smaller than the natural interindividual variability of renal perfusion (SD = +/- 25%). Perfusion in the cortex was approximately 3-4 times higher compared to the medulla. Considering the relatively high resolution of 2x2x10 mm3, the ability to quantify perfusion, and the lack of ionizing radiation and contrast media, this technique should prove useful in diagnosing renal pathologies that are associated with reductions in tissue perfusion.

Adult↗

PubMedDate not supplied

[Study of the structure of erythrocyte membrane in spontaneous genetic hypertension using the spin-label method].

An analysis of ESR spectra of maleimid spin-labeled erythrocyte membranes of spontaneously hypertensive rats of SHR line and normotensive rats of the control line WKY showed differences in the structure of membrane proteins in the norm and pathology. These differences were compared with the differences between the erythrocyte membranes of SHR and WKY, found earlier by fluorescent probe method. An important role of membrane peripheric proteins in the appearance of the above differences in suggested.

Animals↗

PubMed1983

Incorporation of ganglioside analogues into fibroblast cell membranes. A spin-label study.

The uptake of ganglioside analogues by a permanent mouse fibroblast cell line has been studied by radio-tracer techniques and ESR spectroscopy with 3H- and nitroxide-labeled compounds. Analogues of GM1, GM2, and GM3 monosialogangliosides and of GD1a and GD3 disialogangliosides were synthesized. The spin-label group was situated on the 5-, 9-, or 13-carbon atom of the C18 fatty acid chain, and the 3H label was in the carbohydrate moiety. Part of the ganglioside associated with the cells could be removed by trypsin treatment and was shown to consist of ganglioside micelles attached to the cell surface. The trypsin-resistant component displayed characteristic anisotropic ESR spectra which closely resembled those of the same spin-labeled analogues at low dilution in liposomes prepared from the extracted cell lipids. The flexibility gradient, polarity profile, and temperature dependence displayed by the spectra were similar to those found for fluid phospholipid bilayer model membranes, and the high effective order parameters suggested a location in the cell plasma membrane. Similar results were obtained for all the different ganglioside analogues, indicating a common anchoring region in the hydrophobic interior of the membrane. Under the incubation conditions used the amount of trypsin-resistant ganglioside analogue taken up by the cells was about 15 nmol/mg of cellular protein, irrespective of the nature of the oligosaccharide moiety. By use of the natural ganglioside [3H]GM3, the trypsin-resistant uptake was about 19 nmol/mg of cellular protein. Although these amounts are quite similar, the uptake kinetics differed between the true ganglioside GM3 and the ganglioside analogues.

Animals↗

PubMed1986

Interaction between calmodulin and five different spin-labelled chlorophenothiazines.

The binding to purified calmodulin of five spin-labelled derivatives of chlorophenothiazine was investigated by e.s.r. spectrometry and by the antagonizing potency on the calmodulin-dependent activation of myosin light chain kinase. The results of a comparative study and the influence of pH and ionic strength on the binding support the occurrence of an electrostatic binding involving the terminal amino group of the side-chain of the chlorophenothiazine. These results are discussed in relation to the specificity of the interaction that holds the antipsychotic drug-calmodulin complex together.

Calcium↗

PubMed1999

First synthesis of a fully active spin-labeled peptide hormone.

For the first time in the electron spin resonance (ESR) and peptide synthesis fields, a fully active spin-labeled peptide hormone was reported. The ESR spectra of this alpha-melanocyte stimulating hormone (alpha-MSH) analogue (acetyl-Toac0-alpha-MSH) where Toac is the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, suggested a pH-independent conformation and a more restricted movement comparatively to the free Toac. Owing to its equivalent biological potency in a skin pigmentation assay as compared to the native alpha-MSH and its unique characteristic (paramagnetic, naturally fluorescent and fully active), this analogue is of great potential for investigation of relevant physiological roles reported for alpha-MSH.

Electron Spin Resonance Spectroscopy↗

PubMed1995

Resolution of three structural states of spin-labeled myosin in contracting muscle.

We have used electron paramagnetic resonance (EPR) spectroscopy to detect ATP- and calcium-induced changes in the structure of spin-labeled myosin heads in glycerinated rabbit psoas muscle fibers in key physiological states. The probe was a nitroxide iodoacetamide derivative attached selectively to myosin SH1 (Cys 707), the conventional EPR spectra of which have been shown to resolve several conformational states of the myosin ATPase cycle, on the basis of nanosecond rotational motion within the protein. Spectra were acquired in rigor and during the steady-state phases of relaxation and isometric contraction. Spectral components corresponding to specific conformational states and biochemical intermediates were detected and assigned by reference to EPR spectra of trapped kinetic intermediates. In the absence of ATP, all of the myosin heads were rigidly attached to the thin filament, and only a single conformation was detected, in which there was no sub-microsecond probe motion. In relaxation, the EPR spectrum resolved two conformations of the myosin head that are distinct from rigor. These structural states were virtually identical to those observed previously for isolated myosin and were assigned to the populations of the M*.ATP and M**.ADP.Pi states. During isometric contraction, the EPR spectrum resolves the same two conformations observed in relaxation, plus a small fraction (20-30%) of heads in the oriented actin-bound conformation that is observed in rigor. This rigor-like component is a calcium-dependent, actin-bound state that may represent force-generating cross-bridges. As the spin label is located near the nucleotide-binding pocket in a region proposed to be pivotal for large-scale force-generating structural changes in myosin, we propose that the observed spectroscopic changes indicate directly the key steps in energy transduction in the molecular motor of contracting muscle.

Adenosine Triphosphate↗

PubMed2000

Lipid chain dynamics in stratum corneum studied by spin label electron paramagnetic resonance.

The lipid chain motions in stratum corneum (SC) membranes have been studied through electron paramagnetic resonance (EPR) spectroscopy of stearic acid spin-labeled at the 5th, 12th and 16th carbon atom positions of the acyl chain. Lipids have been extracted from SC with a series of chloroform/methanol mixtures, in order to compare the molecular dynamics and the thermotropic behavior in intact SC, lipid-depleted SC (containing covalently bound lipids of the corneocyte envelope) and dispersion of extracted SC lipids. The segmental motion of 5- and 12-doxylstearic acid (5- and 12-DSA) and the rotational correlation time of 16-doxylstearic acid (16-DSA) showed that the envelope lipids are more rigid and the extracted lipids are more fluid than the lipids of the intact SC over the range of temperature measured. The lower fluidity observed for the corneocyte envelope, that may be caused mainly due to lipid-protein interactions, suggests a major contribution of this lipid domain to the barrier function of SC. Changes in the activation energy for reorientational diffusion of the 16-DSA spin label showed apparent phase transitions around 54 degrees C, for the three SC samples. Some lipid reorganization may occur in SC above 54 degrees C, in agreement with results reported from studies with several other techniques. This reorganization is sensitive to the presence of the extractable intercellular lipids, being different in the lipid-depleted sample as compared to native SC and lipid dispersion. The results contribute to the understanding of alkyl chain packing and mobility in the SC membranes, which are involved in the mechanisms that control the permeability of different compounds through skin, suggesting an important involvement of the envelope in the skin barrier.

Animals↗

PubMed2002

Comparison of quantitative perfusion imaging using arterial spin labeling at 1.5 and 4.0 Tesla.

High-field arterial spin labeling (ASL) perfusion MRI is appealing because it provides not only increased signal-to-noise ratio (SNR), but also advantages in terms of labeling due to the increased relaxation time T(1) of labeled blood. In the present study, we provide a theoretical framework for the dependence of the ASL signal on the static field strength, followed by experimental validation in which a multislice pulsed ASL (PASL) technique was carried out at 4T and compared with PASL and continuous ASL (CASL) techniques at 1.5T, both in the resting state and during motor activation. The resting-state data showed an SNR ratio of 2.3:1.4:1 in the gray matter and a contrast-to-noise ratio (CNR) of 2.7:1.1:1 between the gray and white matter for the difference perfusion images acquired using 4T PASL, 1.5T CASL, and 1.5T PASL, respectively. However, the functional data acquired using 4T PASL did not show significantly improved sensitivity to motor cortex activation compared with the 1.5T functional data, with reduced fractional perfusion signal change and increased intersubject variability. Possible reasons for these experimental results, including susceptibility effects and physiological noise, are discussed.

Adult↗

PubMed1988

Delivery of nitroxide spin label to cultured cells by liposomes.

The positively charged nitroxide spin label, 2,2,6,6-tetramethyl-piperidine-N-oxyl-4-trimethylammonium (Cat1), was encapsulated in two types of liposomes, phosphatidylserine/phosphatidylcholine (PS/PC) and phosphatidylserine/distearoylphosphatidylcholine/dipalmitoylphosphatidyl choline (PS/DSPC/DPPC). The liposomes were incubated with mouse thymus-bone marrow (TB) cells to study the uptake and metabolism of nitroxides entrapped in liposomes. The effects of temperature, metabolic inhibitors, and fixation of cells were investigated. The results indicate that different mechanisms are involved in the uptake of these two types of liposomes. PS/PC liposomes interact predominantely with the plasma membrane of TB cells and release Cat1 continuously, whereas the majority of PS/DSPC/DPPC liposomes are taken into the cells intact via endocytosis. These findings suggest that it may be possible to deliver nitroxides selectively, either to the membrane of cells or to their interior by manipulating the lipid composition of the liposomes. This study also found that the rate of reduction of Cat1 delivered using liposomes was increased under hypoxic conditions. Thus, the use of liposomes for in vivo delivery of nitroxides has the potential to provide NMR contrast that reflects different metabolic conditions.

Animals↗