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PubMed1980

Effect of cholesterol on human erythrocyte membrane. A spin label study.

The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.

Androstanes↗
PubMed1998

Guanidine hydrochloride unfolding of a transmembrane beta-strand in FepA using site-directed spin labeling.

We have used the electron spin resonance (ESR) site-directed spin-labeling (SDSL) technique to examine the guanidine hydrochloride (Gdn-HCl) induced denaturation of several sites along a transmembrane beta-strand located in the ferric enterobactin receptor, FepA. In addition, we have continued the characterization of the beta-strand previously identified by our group (Klug CS et al., 1997, Biochemistry 36:13027-13033) to extend from the periplasm to the extracellular surface loop in FepA, an integral membrane protein containing a beta-barrel motif comprised of a series of antiparallel beta-strands that is responsible for transport of the iron chelate, ferric enterobactin (FeEnt), across the outer membrane of Escherichia coli and many related enteric bacteria. We have previously shown that a large surface loop in FepA containing the FeEnt binding site denatures independently of the beta-barrel domain (Klug CS et al., 1995, Biochemistry 34:14230-14236). The SDSL approach allows examination of the unfolding at individual residues independent of the global unfolding of the protein. This work shows that sites along the beta-strand that are exposed to the aqueous lumen of the channel denature more rapidly and with higher cooperativity than the surface loop, while sites on the hydrophobic side of the beta-strand undergo a limited degree of noncooperative unfolding and do not fully denature even at high (e.g., 4 M) Gdn-HCl concentrations. We conclude that, in a transmembrane beta-strand, the local environment of a given residue plays a significant role in the loss of structure at each site.

Bacterial Outer Membrane Proteins↗
PubMed1985

Spin-label studies of lipid-protein interactions in (Na+,K+)-ATPase membranes from rectal glands of Squalus acanthias.

Lipid-protein interactions in (Na+,K+)-ATPase-rich membranes from the rectal gland of Squalus acanthias have been studied by using spin-labeled lipids in conjunction with electron spin resonance (ESR) spectroscopy. Lipid-protein associations are revealed by the presence of a second component in the ESR spectra of the membranes in addition to a component which corresponds very closely to the ESR spectra obtained from dispersions of the extracted membrane lipids. This second component corresponds to spin-labeled lipids whose motion is very significantly restricted relative to that of the fluid lipids in the membrane or the lipid extract. A stoichiometry of approximately 66 lipids per 265 000-dalton protein is found for the motionally restricted component of those spin-labeled lipids (e.g., phosphatidylcholine) which show least specificity for the protein. This corresponds approximately to the number of lipids which may be accommodated within the first shell around the alpha 2 beta 2 protein dimer. A selectivity of the various spin-labeled lipids for the motionally restricted component associated with the protein is found in the following order: cardiolipin greater than phosphatidylserine approximately stearic acid greater than or equal to phosphatidic acid greater than phosphatidylglycerol approximately phosphatidylcholine approximately phosphatidylethanolamine approximately androstanol.

Animals↗
PubMed1990

Mapping of collision frequencies for stearic acid spin labels by saturation-recovery electron paramagnetic resonance.

Short pulse saturation-recovery electron paramagnetic resonance methods have been used to measure interactions of 14N:15N stearic acid spin label pairs in multilamellar liposomal dispersions composed of dimyristoyl-phosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC). Pairs consisting of various combinations of [14N]-16-, [14N]-12- or [14N]-5-doxylstearate, and [15N]-16-, [15N]-12-, or [15N]-5-doxylstearate were studied. SR experiments were performed at 27 degrees and 37 degrees C, and recovery signals were analyzed for initial conditions and multiexponential time constants by computer fitting using a damped least-squares approach. The time constants contain combinations of the electron spin lattice relaxation time, Tle, for each member of the spin-label pair, and the Heisenberg exchange rate constant, Kx. Spin-lattice relaxation times for each of the 14N and 15N stearic acid spin labels were determined, and it is noted that Tle for a given 15N-SASL was always slightly greater than that of the corresponding 14N-SASL. From Kx the bimolecular collision frequency was calculated, providing a detailed picture of molecular interactions. For both lipid systems the bimolecular collision rates were ordered as 12:5 less than 16:5 less than 5:5 less than 16:12 less than 12:12 less than 16:16. For all spin-label pairs studied, interaction frequencies were greater in DMPC than in DEPC. For the 16:16, 12:12, and 16:12 pairs, Kx was approximately 30% greater in DMPC than in DEPC, a significantly greater difference than is observed by conventional EPR methods. Further confirmation of the existence of vertical fluctuation of nitroxide moieties that are at the 16- (or 12) position toward the polar surfaces was obtained, with the frequency of 16:5 (or 12:5) interactions ~40% of the 16:16 (or 12:12) interaction frequency. In both DMPC and DEPC, Kx for homogeneous pairs increases as the doxyl moiety is moved further down the alkyl chain (i.e.,5:5 < 12:12 < 16:16), suggesting that steric effects or the local rotational mobility of the nitroxide group influence the frequency at which spin exchange occurs.

Cyclic N-Oxides↗
PubMed1994

Probing conformational states of spin-labeled aspartate aminotransferase by ESR.

Mitochondrial aspartate aminotransferase was selectively labeled with various maleimide-linked nitroxide spin labels at the conformationally sensitive Cys166. The mobility of the spin group was found to increase with increasing length of the spacer between the nitroxide and maleimide moiety. The label with the ethylcarbamoyl group, a spacer of intermediate length, responded sensitively to conformational changes of aspartate aminotransferase. The modification with this label decreased the enzymic activity to 30% of its initial value and increased the affinity for various substrates and inhibitors 5-10-fold. Identical ESR spectra were obtained for the pyridoxal and pyridoxamine form of the enzyme. These spectra are complex, consisting of an isotropic and at least two anisotropic components. The spectral complexity is attributed to different modes of interaction of the spin label with its local protein environment giving rise to different motional states. The same changes in the ESR spectra have been observed upon formation of the adsorption complex of the pyridoxal form with a competitive inhibitor and on formation of covalent intermediates of the transamination reaction. Essentially, the isotropic component is converted to a new anisotropic one as the local environment changes due to a conformational adaptation of aspartate aminotransferase. The ESR data are consistent with an equilibrium between two conformational states of the enzyme but inconsistent with individual protein conformations of the various intermediates of the transamination reaction. The two conformational states may be assigned to the open and closed conformations as defined by X-ray crystallography. In the adsorption complex of the pyridoxal enzyme, and in the covalent intermediates, the two-state equilibrium appears to be shifted towards the closed conformation in which the spin label is more rigidly bound, as also suggested by molecular dynamic simulations of the label modelled into aspartate aminotransferase. In contrast the formation of adsorption complexes between the pyridoxamine form and aspartate or maleate was not accompanied by the same shift of the conformational equilibrium.

Aspartate Aminotransferases↗
PubMed1983

Electron spin resonance studies on the inorganic-anion-transport system of the human red blood cell. Binding of a disulfonatostilbene spin label (NDS-TEMPO) and inhibition of anion transport.

The disulfonatostilbene spin label, NDS-TEMPO, was synthesized (purity over 96%) and the binding of the spin label to human red-cell ghosts was studied. NDS-TEMPO is readily absorbed to the membrane surface. Both pretreatment of the ghosts with FDNB and DIDS and the presence of DNDS completely prevent the binding of NDS-TEMPO to red-cell ghosts. Chloride and sulfate competitively inhibit the binding of NDS-TEMPO. Conversely, NDS-TEMPO is a strong, competitive inhibitor of chloride and of sulfate transport. The dissociation constants of NDS-TEMPO from the ESR studies were in the range 1.0-2.0 microM (pH 7.6, 20 degrees C). The inhibition constants of NDS-TEMPO as obtained from the flux experiments were in the range 0.5-2.5 microM (pH 7.3, 25 degrees C). The close accordance of the NDS-TEMPO dissociation constants from the ESR studies with the NDS-TEMPO inhibition constants from the flux measurements indicate a specific labeling of the inorganic-anion-transport system.

Anions↗
PubMed2004

Association of spin-labeled lipids with beta-barrel proteins from the outer membrane of Escherichia coli.

The interaction of spin-labeled lipids with beta-barrel transmembrane proteins has been studied by the electron spin resonance (ESR) methods developed for alpha-helical integral proteins. The outer membrane protein OmpA and the ferrichrome-iron receptor FhuA from the outer membrane of Escherichia coli were reconstituted in bilayers of dimyristoylphosphatidylglycerol. The ESR spectra from phosphatidylglycerol spin labeled on the 14-C atom of the sn-2 chain contain a second component from motionally restricted lipids contacting the intramembranous surface of the beta-barrel, in addition to that from the fluid bilayer lipids. The stoichiometry of motionally restricted lipids, 11 and 32 lipids/monomer for OmpA and FhuA, respectively, is constant irrespective of the total lipid/protein ratio. It is proportional to the number of transmembrane beta-strands, eight for OmpA and 22 for FhuA, and correlates reasonably well with the intramembranous perimeter of the protein. Spin-labeled lipids with different polar headgroups display a differential selectivity of interaction with the two proteins. The more pronounced pattern of lipid selectivity for FhuA than for OmpA correlates with the preponderance of positively charged residues facing the lipids in the extensions of the beta-sheet and shorter interconnecting loops on the extracellular side of FhuA.

Bacterial Outer Membrane Proteins↗
PubMed2006

Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies.

Ovalbumin (OVA) has been used continuously as the model antigen in numerous studies of immune reactions and antigen processing, very often encapsulated into liposomes. The purpose of this work was to study the possible interactions of spin-labelled OVA and lipids in liposomal membranes using electron spin resonance (ESR) spectroscopy. OVA was covalently spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), characterized and encapsulated into multilamellar, negatively charged liposomes. ESR spectra of this liposomal preparation gave evidence for the interaction of OVA with the lipid bilayers. Such an interaction was also evidenced by the ESR spectra of liposomal preparation containing OVA, where liposomes were spin-labelled with n-doxyl stearic acids. The spin-labelled OVA retains its property to bind specific anti-OVA antibodies, as shown by ESR spectroscopy, but also in ELISA for specific anti-OVA IgG.

Animals↗
PubMed1985

Synthesis and study of spin-labeled nitrosoureas.

For the first time we have synthesized spin-labeled nitrosoureas and have studied their properties--reduction of the iminoxyl group by vitamin C leading to the formation of the corresponding hydroxylamine derivatives and degradation in the presence of an aminoradical, leading to biradicals. The ESR spectra of biradicals in methanol have nine hyperfine resonance lines. The spin-labeled nitrosoureas have shown a high antitumor activity against the L 1210 lymphoid leukemia and P 388 lymphocytic leukemia in BDF1 mice. A study of a broad range of transplantable tumors is in progress.

Animals↗
PubMed1991

Distance changes at the regulatory and catalytic sites on Escherichia coli glutamine synthetase: a spin label study on the effect of substrate(s) binding.

A spin-labeled ATP analogue, 2,2,6,6-tetramethylpiperidine-1-oxyl adenosine triphosphatase (Tempo-ATP) is used to adenylate Escherichia coli glutamine synthetase (L-glutamine: ammonia ligase (ADP-forming), EC 6.3.1.2). The Tempo adenylylated glutamine synthetase (Tempo-GS) exhibits similar catalytic properties, pH profile and inhibitor susceptibility as those of glutamine synthetase adenylylated with normal ATP. Using the spin label on the enzyme as a probe and employing the spin-spin interactions between the label probe and paramagnetic Mn2+, the distances from the nitroxyl moiety of the covalently bound Tempo-AMP to the two Mn2+ binding sites, n1 and n2 were determined. The n1 site is the structural site and n2 is located at the catalytic site. The distances from Mn2+ at n1 and n2 sites to the nitroxyl radical are 19 and 16 A, respectively. Binding of the substrate, L-Glu, causes a protein conformational change which is reflected by the reduction of approximately 2 A for the n1 to Tempo-AMP distance and lengthening of approximately 2 A for the n2 to the Tempo-AMP distance. Addition of ATP to the Tempo-GS/L-Glu complex increases the distance between n1 and Tempo-AMP, and n2 and Tempo-AMP by 4 and 3 A, respectively.

Adenosine Diphosphate↗
PubMedDate not supplied

Spin-labeled 1-alkyl-1-nitrosourea synergists of antitumor antibiotics.

A new method for synthesis of four spin-labeled structural analogues of the antitumor drug 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), using ethyl nitrite for nitrosation of the intermediate spin-labeled ureas has been described. In vitro synergistic effects of 1-ethyl-3-[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)]-1-nitrosourea (3b) on the cytotoxicity of bleomycin and farmorubicin were found in human lymphoid leukemia tumor cells. We measured the tissue distribution of 3b in organ homogenates of C57BL mice by an electron paramagnetic resonance method. The spin-labeled nitrosourea was mainly localized in the lungs. Our results strongly support the development and validation of a new approach for synthesis of less toxic nitrosourea derivatives as potential synergists of antitumor drugs.

Animals↗
PubMed2000

How bradykinin alters the lipid membrane structure: a spin label comparative study with bradykinin fragments and other cations.

Electron spin resonance spectroscopy of several different spin labels was used to comparatively study the interaction of the cationic peptide hormone bradykinin (BK; Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg), and some BK fragments (des-Arg(9)-BK, des-Arg(1)-BK, and Arg-Pro-Pro-Gly-Phe or BK(1-5)), with anionic vesicles of dimyristoyl phosphatidylglycerol (DMPG). For temperatures above the lipid gel-liquid crystal thermal transition (T(m) approximately 20 degrees C), membrane-incorporated spin labels indicated that all peptides (total concentration of 10 mol % relative to lipid) interact with the bilayer, turning the membrane less fluid, both at its surface and center, suggesting a partial penetration of the peptides into the membrane core. However, in the lipid gel phase (t < T(m)), BK was found to display a much stronger interaction with the membrane, decreasing the bilayer fluidity. At temperatures around 15 degrees C the BK-DMPG system was found to present a hysteresis, evinced by the different electron spin resonance spectra yielded upon cooling and heating the sample. System reversibility was found at all other temperatures (0-45 degrees C). That effect could not be assigned to the BK higher concentration at the membrane surface, due to its higher net charge (2(+)) compared to the fragments (1(+)), because ten times more des-Arg(9)-BK (100 mol %) yielded opposite result. Further, that was found to be a result rather different from those elicited by the other cations tested: the monovalent Na(+), the divalent Zn(2+), and the peptide pentalysine. The data presented here are discussed in the light of the different BK and BK fragments biological activities.

Amino Acid Sequence↗
PubMed1985

Spin labelling of sialic acid and galactose residues on lymphocyte plasma membrane: effects of lectins on oligosaccharide dynamics.

To investigate the behaviour of glycoprotein and glycolipid receptors at the lymphocyte cell surface, a spin label probe has been introduced into either sialic acid or galactose residues on lymphocyte plasma membrane, using specific activation of sugars with periodate or galactose oxidase, followed by reductive amination. The extent of membrane labelling could be controlled by varying the mole ratios of reactants used. Chloroform-methanol extraction of the labelled membranes showed that approximately 17% of the label is bound to glycolipids. A large fraction of the spin label could be released from both sialic acid and galactose-labelled membrane by treatment with pronase, indicating attachment to membrane proteins. Rotational correlation times (tau c) for both labelled sialic acid and galactose residues were in the range 10-13 X 10(-10) sec, indicating a reduction in sugar headgroup mobility at the membrane surface. Isolated lymphocyte membrane glycoproteins spin labelled on galactose residues and reassembled into phospholipid bilayer vesicles showed similar motional characteristics. Prolonged incubation of conc. suspensions of labelled membrane resulted in cleavage of the sialic acid-bound (but not the galactose-bound) label. Binding of several lectins to labelled plasma membrane produced significant immobilization of cell surface oligosaccharides while others had no effect. This differential restriction in oligosaccharide motion following lectin binding appears to be at least partly related to the sugar specificity of the lectin. Binding of wheat germ agglutinin and Ricinus communis agglutinin to sialic acid and galactose-labelled membrane respectively produced a dramatic decrease in oligosaccharide mobility which was reversible on addition of the appropriate sugar inhibitor. The concn dependence of lectin-induced spin label immobilization suggested a cooperative interaction between the lectins and their oligosaccharide receptors. Binding of lectins to the lymphocyte cell surface thus seems to have distinct effects on the dynamic state of glycoproteins and glycolipids within the glycocalyx.

Animals↗
PubMed2006

Analysis of nitroxide spin label motion in a protein-protein complex using multiple frequency EPR spectroscopy.

X- and W-band EPR spectra, at room and low temperatures, are reported for nitroxide spin labels attached to cysteine residues selectively introduced into two proteins, the DNase domain of colicin-E9 and its immunity protein, Im9. The dynamics of each site of attachment on the individual proteins and in the tight DNase-Im9 complex have been analysed by computer simulations of the spectra using a model of Brownian dynamics trajectories for the spin label and protein. Ordering potentials have been introduced to describe mobility of labels restricted by the protein domain. Label mobility varies with position from completely immobilised, to motionally restricted and to freely rotating. Bi-modal dynamics of the spin label have been observed for several sites. We show that W-band spectra are particularly useful for detection of anisotropy of spin label motion. On complex formation significant changes are observed in the dynamics of labels at the binding interface region. This work reveals multi-frequency EPR as a sensitive and valuable tool for detecting conformational changes in protein structure and dynamics especially in protein-protein complexes.

Binding Sites↗
PubMed1997

The membrane topology of the fusion peptide region of influenza hemagglutinin determined by spin-labeling EPR.

Hemagglutinin (HA) is a homotrimeric surface glycoprotein of the influenza virus. In infection, it induces membrane fusion between viral and endosomal membranes at low pH. Each monomer consists of the receptor-binding HA1 domain and the membrane-interacting HA2 domain. It has been known that the NH2-terminal region of the HA2 domain, the so-called "fusion peptide", inserts into the target membrane and plays a crucial role in triggering fusion between the viral and endosomal membranes. A major portion of the HA2 domain (FHA2: aa 1 to 127) of influenza virus X-31, including the NH2-terminal fusion peptide region, was expressed in Escherichia coli. Through site-directed mutagenesis, eight cysteine (Cys) mutants in the fusion peptide region of HA2 (A5C, I6C, A7C, G8C, I10C, N12C, G13C, W14C) were generated and modified with a nitroxide spin label. Using spin-labeling electron paramagnetic resonance (EPR) techniques, we investigated the conformation, membrane topology and the local oligomeric state of the fusion peptide region in the membrane. EPR spectra showed that this region is likely to exist as a flexible monomer in the membrane at both neutral and fusogenic pH conditions. In addition, EPR power saturation methods allowed us to measure the depth in the membrane of the spin label at each mutation site. The resulting depth profile is consistent with an alpha-helix tilted approximately 25 degrees from the horizontal plane of the membrane with a maximum depth of 15 A from the phosphate group. The tilt and rotational orientation correlates well with a calculated amphiphilicity of this region.

Amino Acid Sequence↗
PubMed1990

Spin label ESR and 31P-NMR studies of the cubic and inverted hexagonal phases of dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) mixtures.

The thermotropic phase behaviour and chain dynamics of the dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) mixture at pH 4.0 have been studied with 31P-NMR and spin-label ESR. Broadline proton-dipolar decoupled 31P-NMR spectra show that the system undergoes a transition from a lamellar phase to a non-lamellar phase at 47 degrees C. The 31P-NMR spectrum collapses, from a lamellar powder pattern with apparent chemical shift anisotropy delta sigma = -38 ppm below 47 degrees C, to an isotropic spectrum with superimposed hexagonal powder pattern of delta sigma = +13 ppm above 47 degrees C. With increasing temperature, the hexagonal component grows at the expense of the isotropic component, finally transforming to a single hexagonal phase at approximately 70 degrees C. The ESR spectra from both fatty acid and phosphatidylcholine probes spin-labelled in the hydrocarbon chain have been used to study the ordering and mobility of the lipid chains through the lamellar-non lamellar transition. An abrupt increase in the chain mobility observed at approx. 47 degrees C demonstrates that the lamellar-non lamellar phase transition coincides with chain melting. The apparent order parameter and polarity profiles, deduced from the ESR spectra of 11 different chain-labelled positional isomers, demonstrate flexibility and polarity gradients characteristic of a lyotropic liquid crystalline phase. The temperature dependence of the order parameters does not show any changes in the fluid phase, indicating that the conversion from the isotropic (cubic) phase to the inverted hexagonal phase is not accompanied by large changes in chain order. A comparison of the apparent order parameters and polarity profiles of the phosphatidylcholine and fatty acid spin labels indicates that the fatty acid spin labels are intercalated approximately one CH2 unit more deeply in the hydrocarbon region than are the positionally isomeric phosphatidylcholine spin labels.

Chemical Phenomena↗
PubMed1983

Carbon-13 nuclear magnetic resonance study of spin labelled cholesteryl ester in model membranes.

Carbon-13 NMR longitudinal relaxation times for unilamellar vesicles of egg phosphatidylcholine (PC) in aqueous dispersion have been measured following the incorporation of spin labelled cholesteryl palmitate. The spin label induced relaxation rates, 1/T1,SL, for fatty acyl chain carbons show that the C5 segment of the cholesteryl ester acyl chain is located near the C1 and C2 segments of the phospholipid acyl chains. A greater spin label induced enhancement of relaxation rate was observed for the inner vesicle layer than for the outer, and is attributed to a higher ester incorporation and/or tighter lipid packing in the inner layer.

Cholesterol Esters↗