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PubMed2005

New method for determining tie-lines in coexisting membrane phases using spin-label ESR.

A full description of coexisting phases includes their respective compositions, which are provided by the thermodynamic tie-lines. Fluorescence microscopy enables visualization of coexisting lipid phases in giant unilamellar phases, but the composition information is missing. For cholesterol-containing lipid mixtures, knowledge of the compositions of the coexisting phases is important for understanding the nature of "membrane rafts". We propose and demonstrate a new method, based on ESR spectroscopy, for determining tie-lines in regions of two-phase coexistence in a ternary lipid mixture. Over 100 different lipid compositions containing the spin-labeled phospholipid 16-PC in or near the two-phase coexistence region of the liquid-disordered and the gel phases of dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/Chol) were studied to determine five tie-lines, spread over virtually the full range of this coexistence region. The method is based on the facts that (1) along a tie-line the ESR spectrum must be a superposition of the two ESR spectra from the respective single phases at the phase boundaries (connected by the tie-line) in a ratio given by the lever rule; (2) along a tie-line the partition coefficient, K(p), for the spin-label, which is also determined in our method, must be constant. We do find that K(p) for 16-PC is close to unity, but its value depends on the particular tie-line. The coexisting phases in equilibrium are characterized by the K(p) of the spin-label and its respective dynamic parameters obtained from fitting the ESR spectra to dynamical models.

Cholesterol↗
PubMed1976

Ca2+-induced conformational changes of spin-labeled g2 chain bound to myosin and the effect of phosphorylation.

One of the low molecular weight components of myosin, g2, was isolated by alkali treatment of myosin and was chemically modified with a spin label reagent, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. The label on g2 showed a rather weakly immobilized ESR spectrum and it was clearly affected by Ca2+; the half-maximal change was at around pCa 4. The spin-labeled g2 was incorporated into myosin by exchange with the intrinsic g2 of myosin in 0.6 M KSCN or 4 M LiC1. The label on g2 became strongly immobilized on association with myosin. Under the conditions used, ESR spectral change due to Ca2+ occurred at two different concentration ranges, which were as low as pCa 8 and at around pCa 4. Phosphorylated g2 was isolated from myosin after the protein kinase [EC 2.1.1.37]-catalyzed phosphorylation of myosin and it was also modified with the maleimide label. Dephosphorylation of the phosphorylated g2 was performed using E. coli alkaline phosphatase [EC 3.1.3.1]. The effects of Ca2+ on the ESR spectra of phosphorylated and dephosphorylated g2 were investigated on the state associated with myosin. A change in the ESR spectrum from strongly immobilized to weakly immobilized states was observed with both g2 chains on the addition of Ca2+. However, the effective concentration ranges of Ca2+ were quite different; around pCa 4 for the phosphorylated g2 and around pCa 8 for the dephosphorylated g2. The results indicate that g2 undergoes a conformational change at physiological levels of Ca2+ sufficient to saturate troponin, but it does not do so after phosphorylation.

Alkaline Phosphatase↗
PubMed2003

C2 domain of protein kinase C alpha: elucidation of the membrane docking surface by site-directed fluorescence and spin labeling.

The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca(2+)-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase C alpha (PKC alpha) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca(2+) binding loops and an anion binding site on beta-strands 3-4, such that the beta-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca(2+) binding loops and the beta-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKC alpha C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca(2+) activation or membrane docking trigger measurable fluorescence changes. Ca(2+) binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca(2+) binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Phi, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca(2+) binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca(2+) first and third Ca(2+) binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on beta-strands 3-4 lies near the headgroups. The data support a model in which the beta-strands are tilted toward the parallel orientation relative to the membrane surface.

Amino Acid Substitution↗
PubMed1989

Decrease in the fluidity of brush-border membrane vesicles induced by gentamicin. A spin-labeling study.

In our previous paper (Horio et al., Biochim Biophys Acta 858: 153-160, 1986), we reported that the addition of gentamicin in vitro to rabbit renal brush-border membrane vesicles decreases the apparent Vmax of Na+-dependent D-glucose transport without affecting the apparent Km. In the present study, we investigated the effects of gentamicin on the physical state of spin-labeled rabbit renal brush-border membranes, using electron spin resonance spectrometry. Brush-border membrane vesicles were prepared from outer cortex (mainly contains early proximal tubule) and outer medulla (containing primarily late proximal tubule), and the gentamicin toxicities in both preparations were compared. Significant decreases were observed in the membrane fluidity of 5 mM gentamicin-treated brush-border membranes. The fluidity of outer cortical brush-border membranes was affected at both 25 degrees and 35 degrees, whereas that of outer medullary membranes was affected only at 35 degrees. Two different stearic acid spin labels revealed that gentamicin affected the fluidity only in the superficial region of the membranes. We also demonstrated that the gentamicin-induced decreases in Na+-dependent D-glucose transport and in the membrane fluidity were recovered by washing gentamicin-treated brush-border membranes. We suggest that gentamicin binds to the superficial region of brush-border membranes and inhibits Na+-dependent D-glucose transport across brush-border membranes through the decrease in the membrane fluidity.

Animals↗
PubMed1977

Binding studies of a spin-labelled oxidized coenzyme to bovine-liver glutamate dehydrogenase.

NAD+ with a nitroxide piperidine ring linked to the NH2 group of the adenine possesses full coenzymatic activity with glutamate dehydrogenase. Electron spin resonance spectra in the presence of glutamate dehydrogenase show mixtures of free and strongly immobilized spin-label. Binding studies in phosphate buffer demonstrate: (a) weak binary binding to the enzyme with a dissociation constant in the order of 2mM;(b) an indication for negative cooperativity or different sites for binding to enzyme-2-oxoglutarate, with dissociation constants in the order of 20--250muM; (c) similar but much weaker binding to enzyme-2-oxoglutarate-ADP; (d) a strong positive cooperative binding to enzyme-2-oxoglutarate-GTP, dependent on the enzyme concentration. Binding of phosphate to the enzyme with a Kd of about 20 mM or binding of pyrophosphate or tripolyphosphate with a Dd of about 2.5 mM enhances the binding of spin-labelled NAD+ in the presence of 2-oxoglutarate. There is evidence that the binding sites for these phosphates coincide with phosphate binding subsites of GTP.

Animals↗
PubMed1993

Orientation and rotational dynamics of spin-labeled phalloidin bound to actin in muscle fibers.

We have used electron paramagnetic resonance spectroscopy (EPR) to investigate the orientational distribution of actin in thin filaments of glycerinated muscle fibers in rigor, relaxation, and contraction. A spin-labeled derivative of a mushroom toxin, phalloidin (PHSL), was bound to actin in the muscle fibers (PHSL-fibers). The EPR spectrum of unoriented PHSL-labeled myofibrils consisted of three sharp lines with a splitting between the outer extrema (2T parallel') of 42.8 +/- 0.1 G, indicating that the spin labels undergo restricted nanosecond rotational motion within an estimated half-cone angle of 76 degrees. When the PHSL-fiber bundle was oriented parallel to the magnetic field, the splitting between the zero-crossing points (2T') was 42.7 +/- 0.1 G. When the fiber bundle was perpendicular to the magnetic field, 2T' decreased to 34.5 +/- 0.2 G. This anisotropy shows that the motion of the probe is restricted in orientation by its binding site on actin, so that the EPR spectrum of PHSL-fiber bundles would be sensitive to small changes in the mean axial orientation of the PHSL-actin interface. No differences in the EPR spectra were observed in fibers during rigor, relaxation, or contraction, indicating that the mean axial orientation of the PHSL binding site changes by less than 5 degrees, and that the amplitude of nanosecond probe rotational motion, which should be quite sensitive to the local environment of the phalloidin, changes by no more than 1 degree.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1990

Sterically-hindered hydroxylamines as bioactive spin labels.

The use of sterically hindered hydroxylamines for the regulation of free radical reactions in biological systems and for the pharmacological correction of pathological conditions is described. They are shown to possess a number of advantages over nitroxyl radicals. They are more soluble in water, are less toxic and are easily oxidized to nitroxyl radicals in aqueous solutions. Hindered hydroxylamines can be also used for the study of pharmacokinetics of bioactive spin labels by the EPR technique. Pharmacokinetic parameters for spin-labeled analogues of tetronal are evaluated. Sulfur-containing hindered hydroxylamines are effective bioantioxidants, inhibiting efficiently the LPO of the microsomal fraction of liver and ADP- and thrombin induced plasma platelet aggregation. They increase also the survival of animals under ischemic shock. A cyclic mechanism for its antioxidative action is suggested. Recent data on the influence of the nitroxyl in equilibrium hydroxylamine moiety on bio-activity are summarized.

Animals↗
PubMed2004

Motion of spin label side chains in cellular retinol-binding protein: correlation with structure and nearest-neighbor interactions in an antiparallel beta-sheet.

A goal in the development of site-directed spin labeling in proteins is to correlate the motion of a nitroxide side chain with local structure, interactions, and dynamics. Significant progress toward this goal has been made using alpha-helical proteins of known structure, and the present study is the first step in a similar exploration of a beta-sheet protein, cellular retinol-binding protein (CRBP). Nitroxide side chains were introduced along both interior and edge strands. At sites in interior strands, the side-chain motion is strongly influenced by interactions with side chains of neighboring strands, giving rise to a rich variety of dynamic modes (weakly ordered, strongly ordered, immobilized) and complex electron paramagnetic resonance spectra that are modulated by strand twist. The interactions giving rise to the dynamic modes are explored using mutagenesis, and the results demonstrate the particular importance of the non-hydrogen-bonded neighbor residue in giving rise to highly ordered states. Along edge strands of the beta-sheet, the motion of the side chain is simple and weakly ordered, resembling that at solvent-exposed surfaces of an alpha-helix. A simple working model is proposed that can account for the wide variety of dynamic modes encountered. Collectively, the results suggest that the nitroxide side chain is an effective probe of side-chain interactions, and that site-directed spin labeling should be a powerful means of monitoring conformational changes that involve changes in beta-sheet topology.

Animals↗
PubMed1998

Cytochrome c-induced increase of motionally restricted lipid in reconstituted cytochrome c oxidase membranes, revealed by spin-label ESR spectroscopy.

Cytochrome c oxidase isolated from beef heart mitochondria was reconstituted in bilayer membranes of the anionic lipid dimyristoylphosphatidylglycerol (DMPG) with varying enzyme/DMPG ratio. Lipid-protein interactions in the reconstituted membrane complexes were studied in the presence and absence of saturating amounts of bound cytochrome c, by both chemical binding assays and spin-label ESR spectroscopy. The ESR spectra from a phosphatidylglycerol probe spin-labeled on C-14 of the sn-2 chain revealed two distinct lipid populations differing in their rotational mobility. The stoichiometry of lipids that were restricted in their rotational motion by direct interaction with the integral protein was 50-60 lipids/cytochrome c oxidase monomer, in the absence of cytochrome c, independent of the total lipid/protein ratio. Cytochrome c alone did not induce a motionally restricted population in the lipid ESR spectra, when bound to bilayers of negatively charged DMPG alone, in the fluid phase (at 36 degreesC). However, the motionally restricted lipid population associated with reconstituted cytochrome c oxidase/DMPG membranes increased on binding cytochrome c, indicating structural/dynamic changes taking place in the membrane. Depending on the DMPG/cytochrome c oxidase ratio, apparent stoichiometries of up to 115 motionally restricted lipid molecules/cytochrome c oxidase monomer were found, when saturating amounts of cytochrome c were bound. Under these conditions, cytochrome c binds to approximately 9 negatively charged DMPG molecules, independent of the cytochrome c oxidase content in the reconstituted system. A likely explanation for these results is that the surface binding of cytochrome c propagates the motional restriction of the lipid chains beyond the first boundary shell of cytochrome c oxidase, possibly creating microscopic in-plane domains.

Animals↗
PubMed1986

Murine in vivo L-band ESR spin-label oximetry with a loop-gap resonator.

Small mice (approximately 20 g) were anesthetized and placed in a loop-gap resonator of diameter 25 mm resonating at 1.1 GHz. An oxygen-permeable capsule containing 2.5 X 10(-2) M perdeutero 15N TEMPONE (1-oxyl-2,2,6,6-tetramethyl-4-piperidone) in light paraffin oil was implanted in the peritoneal cavity. Electron spin resonance (ESR) spectra from the capsule are sensitive to the concentration of dissolved oxygen and calibration curves are given. The oxygen concentration was found to rise from a value close to zero when the animal breathes air to a level of 220 microM when the animal breaths pure oxygen. It is speculated that this surprisingly high level is related to the effect of the anesthetic on the cardiovascular system. Encapsulation provides a barrier to spin-label reductants and to paramagnetic metal ions that might confound the spin-label oximetric measurements.

Animals↗
PubMed2006

Quantification of cerebral arterial blood volume using arterial spin labeling with intravoxel incoherent motion-sensitive gradients.

Quantification of cerebral arterial blood volume (CBVa) is important for understanding vascular regulation. To enable measurement of CBVa with diffusion-weighted (DW) arterial spin labeling (ASL), a theoretical framework was developed using the effects of intravoxel incoherent motion (IVIM). The pseudo-diffusion coefficient (D*) in the IVIM model was evaluated at 9.4 T in DW-ASL of rat brain under isoflurane anesthesia by variations of both post-labeling delay (w) and magnetization transfer ratio (MTR). D* and its volume fraction decreased at values of w>or=0.3 s, and the normalized apparent diffusion coefficient (ADC) increased with MTR, suggesting that D* is closely correlated with CBVa. Thus, the difference between ASL measurements with and without DW gradients is related to CBVa. The CBVa values measured by this approach were compared with values obtained using the modulation of tissue and vessel (MOTIVE) technique with ASL, which varies MT levels without changing spin labeling efficiency. CBVa values from both methods were highly correlated. The measured CBVa values were linearly correlated with cerebral blood flow (CBF) for a PaCO2 range of 25-50 mmHg; DeltaCBVa (ml/100 g)=0.007 (min-1)xDeltaCBF (ml/100 g/min). The DW-ASL approach is simple and easy to implement for human and animal CBVa studies.

Algorithms↗
PubMed2001

Fmoc-POAC: [(9-fluorenylmethyloxycarbonyl)-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid]: a novel protected spin labeled beta-amino acid for peptide and protein chemistry.

The stable free radical 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) is the only spin labeled amino acid that has been used to date to successfully label peptide sequences for structural studies. However, severe difficulty in coupling the subsequent amino acid has been the most serious shortcoming of this paramagnetic marker. This problem stems from the low nucleophilicity of TOAC's amine group towards the acylation reaction during peptide chain elongation. The present report introduces the alternative beta-amino acid 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid (POAC), potentially useful in peptide and protein chemistry. Investigations aimed at addressing the stereochemistry of this cyclic molecule through X-ray diffraction measurements of crystalline and bulk samples revealed that it consists only of the trans conformer. The 9-fluorenylmethyloxycarbonyl group (Fmoc) was chosen for temporary protection of the POAC amine function, allowing insertion of the probe at any position in a peptide sequence. The vasoactive octapeptide angiotensin II (All, DRVYIHPF) was synthesized by replacing Pro7 with POAC. The reaction of Fmoc-POAC with the peptidyl-resin occurred smoothly, and the coupling of the subsequent amino acid showed a much faster reaction when compared with TOAC. POAC7-AII was obtained in good yield, demonstrating that, in addition to TOAC, POAC is a convenient amino acid for the synthesis of spin labeled peptide analogues. The present findings open the possibility of a wide range of chemical and biological applications for this novel beta-amino acid derivative, including structural investigations involving its differentiated bend-inducing characteristics.

Amino Acids↗
PubMed1995

Analysis of saturation transfer electron paramagnetic resonance spectra of a spin-labeled integral membrane protein, band 3, in terms of the uniaxial rotational diffusion model.

Algorithms have been developed for the calculation of saturation transfer electron paramagnetic resonance (ST-EPR) spectra of a nitroxide spin-label assuming uniaxial rotational diffusion, a model that is frequently used to describe the global rotational dynamics of large integral membrane proteins. One algorithm explicitly includes terms describing Zeeman overmodulation effects, whereas the second more rapid algorithm treats these effects approximately using modified electron spin-lattice and spin-spin relaxation times. Simulations are presented to demonstrate the sensitivity of X-band ST-EPR spectra to the rate of uniaxial rotational diffusion and the orientation of the nitroxide probe with respect to the diffusion axis. Results obtained by using the algorithms presented, which are based on the transition-rate formalism, are in close agreement with those obtained by using an eigenfunction expansion approach. The effects of various approximations used in the simulation algorithms are considered in detail. Optimizing the transition-rate formalism to model uniaxial rotational diffusion results in over an order of magnitude reduction in computation time while allowing treatment of nonaxial A- and g-tensors. The algorithms presented here are used to perform nonlinear least-squares analyses of ST-EPR spectra of the anion exchange protein of the human erythrocyte membrane, band 3, which has been affinity spin-labeled with a recently developed dihydrostilbene disulfonate derivative, [15N,2H13]-SL-H2DADS-MAL. These results suggest that all copies of band 3 present in intact erythrocytes undergo rotational diffusion about the membrane normal axis at a rate consistent with a band 3 dimer.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2005

Probing the hydroxyl radical-mediated reactivity of peroxynitrite by a spin-labeling fluorophore.

The decomposition of peroxynitrite at physiological pH yielded a hydroxyl radical, which reacted rapidly with dimethyl sulfoxide (DMSO) to produce a methyl radical (*CH3), which was then trapped by a spin-label fluorophore nitroxide-linked naphthalene (NTEMPO), a carbon-centered radical probe with a low fluorescence intensity, and transformed to a stable diamagnetic O-alkoxyamine, a high-fluorescence compound. The fluorescence increment was proportional to the concentration of the hydroxyl radical, and then to the concentration of peroxynitrite. NTEMPO therefore was demonstrated to be capable of detecting hydroxyl radicals generated from peroxynitrite, and the method was proved to be simple and sensitive. The hydroxyl radical-mediated reactivities of peroxynitrite to several amino acids, such as tyrosine, phenylalanine and histidine, were then evaluated by the spin-labeling fluorophore NTEMPO at pH 7.4 and, 37 degrees C. The obtained data were in good agreement with the reference values, respectively.

Carbon Dioxide↗
PubMed2002

Polarity contributions to hyperfine splittings of hydrogen-bonded nitroxides--the microenvironment of spin labels.

A self-consistent treatment of reaction field effects on isotropic (14)N hyperfine coupling constants of nitroxide spin labels in mixtures of polar and apolar solvents is given based on the Onsager approach. It is shown that this works reasonably well for mixtures of water or methanol with dioxane, far better than do conventional approaches using the Clausius-Mossotti relation. Association constants, K(A,h), for hydrogen bonding of protic solvents to nitroxides are derived in this way from published EPR data. A value of K(A,h) approximately 1.0 M x (-1) is argued to be reasonable for water in a hydrophobic environment. Data from spin-labelled lipids can then be used to estimate effective water concentrations in biological membranes.

Journal Article↗
PubMed1997

Site-directed spin-labeling study of the structure and subunit interactions along a conserved sequence in the alpha-crystallin domain of heat-shock protein 27. Evidence of a conserved subunit interface.

Site-directed spin-labeling (SDSL) was used to investigate the secondary structure, solvent accessibility, and tertiary and quaternary interactions along the sequence located between residues 133 and 144 in the alpha-crystallin domain of human heat-shock protein 27 (HSP 27). The sequence is conserved among mammalian sHSP and shows similarity to the region of highest homology between alpha A- and alpha B-crystallins. Eleven sequential single cysteine mutants were prepared and reacted with a sulfhydryl-specific spin-label. The accessibilities of attached nitroxide side chains to a paramagnetic probe in the aqueous solution were determined. Spectral line shapes were analyzed in terms of side-chain mobility and spatial proximity to nearby nitroxides. The sequence-specific mobilities and accessibilities varied with a period of 2, consistent with the presence of a beta-strand along the sequence. At even sites, the nitroxide environment is highly ordered with virtually no accessibility to the hydrophilic probe, indicating that one face of the strand is buried. Furthermore, spin-spin interactions between nitroxides in the oligomeric structure strongly suggest that equivalent strands from different subunits are in close spatial proximity. These structural characteristics are remarkably similar to those of the equivalent sequence in alpha A-crystallin [Berengian, A. R., Bova, M. P., and Mchaourab, H. S. (1997) Biochemistry 36, 9951-9957]. In both proteins, a beta-strand spans the sequence and is located at a subunit interface, indicating that one set of interactions between subunits, and its associated symmetry, is conserved. This is the first report of sequence-specific structural similarity between alpha A-crystallin and HSP 27 and the first identification of a conserved secondary structural element in the alpha-crystallin domain.

Amino Acid Sequence↗
PubMed2005

High-field spin-label EPR of lipid membranes.

High-field EPR of spin-labelled lipid chains has proved to be an extremely productive means for biophysical investigations of phospholipid bilayer membranes. Results on the following three topics are reviewed: 1. Non-axial ordering of lipid chains in cholesterol-containing membranes; 2. Transmembrane polarity profiles and water penetration in lipid membranes; 3. Role of HF-EPR in multi-frequency spectral simulations to investigate lipid-chain dynamics in membranes. These results are obtained from phosphatidylcholine spin probes with the nitroxide systematically stepped down the sn-2 chain, in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine (DMPC)-containing cholesterol.

Cholesterol↗
PubMed1980

Spin-label study of histone H1-DNA interaction. Comparative properties of the central part of the molecule and the N and C-amino tails.

Covalent spin labelling of lysine and arginine residues in histone H1 under specified conditions allows the study of histone-DNA interaction. When the labelled histone is free in solution, the electron spin resonance spectrum is characteristic of a label moving in a viscous medium while the label becomes fully immobilized upon the interaction of histone and DNA. This property is used to study histone-DNA interactions under various conditions of ionic strength and thermal denaturation. On the other hand, spin label probes in the globular part of the molecule shows no strong immobilization upon binding to linear DNA, suggesting a special mechanism for the binding of histone H1 to chromatin.

Animals↗