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At least 775 records · Page 43Linked to original sources
PubMed2001

Specific spin labelling of the sugar-H(+) symporter, GalP, in cell membranes of Escherichia coli: site mobility and overall rotational diffusion of the protein.

The D-galactose-H(+) symport protein (GalP) of Escherichia coli is a homologue of the human glucose transport protein, GLUT1. After amplified expression of the GalP transporter in E. coli, other membrane proteins were prereacted with N-ethylmaleimide in the presence of excess D-galactose to protect GalP. Inner membranes were then specifically spin labelled on Cys(374) of GalP with 4-maleimide-2,2,6,6-tetramethylpiperidine-1-oxyl. The electron paramagnetic resonance (EPR) spectra are characteristic of a single labelling site in which the mobility of the spin label is very highly constrained. This is confirmed with other nitroxyl spin labels, which are derivatives of iodoacetamide and indanedione. Saturation transfer EPR spectra indicate that the overall rotation of the GalP protein in the membrane is slow at low temperatures (approx. 2 degrees C), but considerably more rapid and highly anisotropic at physiological temperatures. The rate of rotation about the membrane normal at 37 degrees C is consistent with predictions for a 12-transmembrane helix assembly that is less than closely packed.

Amino Acid Sequence↗
PubMed1983

Intramolecular motility in anti-Dnp antibodies and in their Fab fragments. EPR spectra of the complexes with a spin-labelled hapten in H2O-D2O mixtures at various temperatures.

To study intramolecular motility, the binding sites of the antibodies or Fab fragments were occupied by the spin-labelled hapten Dnp-NO. EPR spectra of the complexes were recorded under various conditions, in particular variable viscosity, and the correlation times of the antibody or its fragment were calculated. Intramolecular motility decreases, i.e. correlation time increases, with temperature in the range from 5 degree C to 30 degrees C. This anomalous dependence points to the important role of hydrophobic bonds in the interactions between domains of the antibody molecule. Substitution of a fraction of H2O by D2O (1 to 13%) is manifested by the increase in correlation time only at 5 degrees C, a maximal effect is already obtained at about 3% D2O. At 20 degrees C or in the presence of perturbants of the hydration shell such as 0.5 M sodium chloride, 1 M urea or 10% butanol, D2O has no appreciable effect. EPR spectra made it possible to study the gradual immobilization of the bound spin-labelled hapten at temperatures lower than 0 degrees C. Water in the vicinity of the bound spin-labelled hapten freezes in three discrete steps, -8 to -13 degrees C, -20 to -40 degrees C, and -40 to -80 degrees C. D2O influences the first and second step so that it shifts the transition to higher temperatures. The results contribute to our understanding of the fine structure of the hydration shell and water in the interdomain space of the antibody molecule.

Animals↗
PubMed1991

EPR studies on the influence of chain length on the segmental motion of spin-labelled fatty acids in dimyristoylphosphatidylcholine bilayers.

The rotational dynamics of spin-labelled fatty acids of different chainlengths (9, 10, 12, 14, 16 and 18 C-atoms) and different positions of labelling (5-C, 6-C and 7-C) have been studied in dimyristoylphosphatidylcholine bilayers using EPR spectroscopy. The segmental flexibility at a given label position is found to vary considerably with the length of the lipid chain, when this is less than that of the dimyristoylphosphatidylcholine host lipid. For both the charged and protonated forms of labelled fatty acids with chainlengths of 9, 10, and 12 C-atoms, the spectral anisotropy decreases steadily with decreasing chainlength in fluid phase bilayers. The differences become especially pronounced at the 7-C position of caprylic acid and the 6-C position of nonanoic acid, where the label is located close to the terminal methyl end of the chain. An unusually high degree of motional freedom is found for both these spin-labels, even in gel phase bilayers. There is relatively little effect of chainlength of the labelled fatty acid when this is longer or comparable to that of the host lipid (i.e., for fatty acid chainlengths of 18, 16 and 14 C-atoms), except if the label position is close to the terminal methyl end of the chain. The implications for the heterogeneous lipid chain composition in biological membranes are discussed.

Dimyristoylphosphatidylcholine↗
PubMed1985

Spin label studies of the essential sulfhydryl group environment in chicken liver fructose-1,6-bisphosphatase.

The local environment of the essential sulfhydryl groups in chicken liver fructose-1,6-bisphosphatase has been investigated by ESR techniques using a series of iodoacetamide spin labels, varying in chain length between the iodoacetate and nitroxide free radical group. The ESR spectrum of spin-labeled chicken liver fructose-1,6-bisphosphatase showed that the sites of labeling were highly immunobilized when the enzyme was chemically modified by spin label iodoacetate, suggesting that the sulfhydryl groups of the protein are in a small, confined environment. From the change in the ESR spectra of these nitroxides as a function of chain length, we conclude that the sulfhydryl group is located in a cleft approx. 10.5A in depth.

Animals↗
PubMed1977

Selective enzymatic radioactive and spin labelling of phospholipids in biological membranes: application to study of temperature-induced changes of microsomal phosphatidylinositols and mitochondrial polyglycerophosphatides.

A new method for the covalent radioactive and spin labelling of lipids within isolated biological membranes has been described in detail and applied to studies of temperature-induced changes of microsomal and mitochondrial membranes. The method is based on the enzymatic use of radioactive substrates carrying covalently bound doxyl derivatives of stearic acid in the biosynthesis of phospholipids in isolated membranes. Radioactive-and spin-labelled lipids bound to the microsomal and mitochondrial membranes were then used as internal probes in monitoring temperature-induced changes of these membranes. Since the analysis of isolated radioactive-and spin-labelled lipids revealed the exact composition of membrane-bound labelled lipids, specific temperature-induced changes were correlated with specific lipids of examined membranes. Phosphatidylinositol of microsomal membranes and polyglycerophosphatides (phosphatidyl-glycerol and cardiolipin) of mitochondrial and inner mitochondrial membranes were found to be involved in the apparent formation of lipid clusters at around 20-30 degrees C. Cardiolipin was found to be involved in the fluidization of inner mitochondrial membranes. These findings are discussed in view of the present state of knowledge of the organization of lipids in biological membranes.

Animals↗
PubMed1989

[Use of electron paramagnetic resonance spectroscopy spin labels in studying the properties of biological membranes].

Electron paramagnetic resonance (EPR) spectroscopy of spin labels has become a widely spread method in biochemistry, molecular biology, and medicine. After a brief view on the structure and functions of biological membranes, basic principles of EPR and particularly of the spin labeling technique are presented. Applications of this technique are demonstrated on the study of various properties of biological membranes, such as phase transition of lipids, properties of different membrane parts, permeability of membranes, inner volumes of liposomes and cells, membrane potentials, lateral diffusion, lipid-protein interaction, and determination of oxygen concentration. As an example the results of a study concerning the effect of chlorpromazine and its derivatives on the dynamics and lipid-protein interaction in synaptosomes are presented.

Animals↗
PubMed1981

Structural and motional changes in glyceraldehyde-3-phosphate dehydrogenase upon binding to the band-3 protein of the erythrocyte membrane examined with [15N,2H]maleimide spin label and electron paramagnetic resonance.

Binding of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase [GAPDHase; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating EC 1.2.1.12], to the cytoplasmic segment of band-3 protein in the erythrocyte (RBC) membrane has been examined by electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopies. GAPDHase, which was isolated from rabbit muscle and labeled with the resolution-enhancing deuterated N-(15N-1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide spin label ([15N,2H]MSL), showed the same binding specificity for the transmembrane band-3 protein of human erythrocyte membranes as reported for unlabeled GAPDHase from human RBC. Experimental EPR lineshapes from soluble and membrane-bound enzymes were analyzed by direct stimulation of spectra and indicated a structural alteration of the bound GAPDHase in the vicinity of the spin label, which was attached covalently to the active-site cysteine-149 residue. A rigorous theoretical analysis of the ST-EPR spectra of soluble and membrane-bound enzyme is presented and utilized in conjunction with model system analysis to demonstrate that the motion of membrane-bound GAPDHase could be characterized by an effective isotropic rotational correlation time of 20 microseconds. This indicated that the GAPDHase--band-4 complex exhibits motional freedom relative to the membrane-spanning segment of the band-3 protein or the RBC. The double substituted spin label [15N,2H]MSL affords gains in sensitivity and resolution that permit studies of membrane-bound enzymes at physiological levels and quantitative simulations of the EPR and ST-EPR lineshapes with reasonable computation times.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1986

Micellar formation of spin-labeled fatty acyl derivatives of lipophilic muramyl dipeptides and their incorporation into liposomal membranes.

A lipophilic muramyl dipeptide (MDP) with a nitroxide moiety in its acyl chain (SL-MDP) and its N-methyl derivative (SL-methyl MDP) were synthesized. The SL-MDPs formed micelles (cmc, 0.1-0.3 mM). The ESR spectra of the SL-MDPs in phosphatidylcholine (PC) liposomes at 25 degrees C consisted of an anisotropic signal and three sharp lines, indicating that both SL-MDPs partitioned between membranes and aqueous phase. The amounts of the SL-MDPs in membranes depended on the phospholipid species and the cholesterol (Chol) content, but no appreciable difference was observed between SL-MDPs. The SL-MDPs partitioned well at 25 degrees C into egg yolk PC liposomes but not into pure dipalmitoylphosphatidylcholine (DPPC), suggesting that the incorporation may be related to the membrane fluidity. Chol enhanced the incorporation into both phospholipids. The mobilities of the SL-MDPs in the membranes were less than that of the corresponding spin-labeled fatty acid. Comparison of the mobilities among SL-MDPs, spin-labeled ganglioside and spin-labeled galactosylceramide showed that the hydrophilicity of the polar group may influence the immobilization of their acyl chains.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1986

Spin-labeling of influenza virus hemagglutinin permits analysis of the conformational change at low pH and its inhibition by antibody.

To study the conformational changes in the hemagglutinin (HA) molecule of A/seal/Mass/1/80 (H7N7) (Seal) influenza virus at low pH, a spin-labeling method was used. This method also permits study of antibody interaction with the HA. A synthetic nitroxide compound was used for spin-labeling of tyrosine residues of the isolated HA molecule. Electron spin resonance (ESR) spectra of the spin-labeled HA at various pH values indicated that a conformational transition occurred under acidic conditions, and around pH 5.8 the HA molecule has maximal flexibility. Since virus-induced hemolysis occurs optimally at pH 5.8-5.9, the HA molecule in the maximally flexible conformation is considered to mediate membrane fusion. The ESR spectra of the antibody-bound HA at various pH values revealed that monoclonal antibodies to different regions on the molecule may inhibit the conformational change by different modes. One antibody inhibited the changes in the HA that resulted in flexibility, while the other did not. These results support the assumption that monoclonal antibodies, which failed to inhibit hemagglutination of the virus yet neutralized viral infectivity, inhibited the fusion step in the viral replication process by interfering with a low pH-induced conformational change in the HA molecule (Kida, H., Webster, R.G. and Yanagawa, R. (1983) Arch. Virol. 76, 91-99).

Animals↗
PubMed1979

Synthesis and biological properties of N2-substituted spin-labeled analogues of actinomycin D.

We have synthesized N2-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)]actinomycin D And the related 1,2-diaminoethane and 1,3-diaminopropane derivatives and evaluated their biological properties. Binding studies with the spin-labeled actinomycin D analogues and DNA were carried out by using circular dichroism, electron spin resonance, and thermal denaturation. These studies have suggested that the derivatives bind to DNA and that their DNA-binding modes are similar but not identical. Spin-labeled actinomycin D derivatives were less potent in inhibiting Escherichia coli DNA-dependent RNA polymerase reaction than actinomycin D and were less toxic to L1210 cells in vitro than the parent compound. Spin-labeled actinomycin D derivatives were more common than the parent compounds against P-388 leukemia cells in vitro with little or no toxicity.

Animals↗
PubMed2005

Combining high-field EPR with site-directed spin labeling reveals unique information on proteins in action.

In the last decade, joint efforts of biologists, chemists and physicists have helped in understanding the dominant factors determining specificity and directionality of transmembrane transfer processes in proteins. In this endeavor, electron paramagnetic resonance (EPR) spectroscopy has played an important role. Characteristic examples of such determining factors are hydrogen-bonding patterns and polarity effects of the microenvironment of protein sites involved in the transfer process. These factors may undergo characteristic changes during the reaction and, thereby, control the efficiency of biological processes, e.g. light-induced electron and proton transfer across photosynthetic membranes or ion-channel formation of bacterial toxins. In case the transfer process does not involve stable or transient paramagnetic species or states, site-directed spin labeling with suitable nitroxide radicals still allows EPR techniques to be used for studying structure and conformational dynamics of the proteins in action. By combining site-directed spin labeling with high-field/high-frequency EPR, unique information on the proteins is revealed, which is complementary to that of X-ray crystallography, solid-state NMR, FRET, fast infrared and optical spectroscopic techniques. The main object of this publication is twofold: (i) to review our recent spin-label high-field EPR work on the bacteriorhodopsin light-driven proton pump from Halobacterium salinarium and the Colicin A ion-channel forming bacterial toxin produced in Escherichia coli, (ii) to report on novel high-field EPR experiments for probing site-specific pK(a) values in protein systems by means of pH-sensitive nitroxide spin labels. Taking advantage of the improved spectral and temporal resolution of high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the transient intermediates of the proteins in biological action is obtained. These intermediates can be observed and characterized while staying in their working states on biologically relevant timescales. The paper concludes with an outlook of ongoing high-field EPR experiments on site-specific protein mutants in our laboratories at FU Berlin and Osnabrück.

Bacteriorhodopsins↗
PubMed1976

An ESR study of the anchoring of spin-labeled stearic acid in lecithin multilayers.

In egg lecithin-water lamellar phases, spin-labeled stearic acid gives two superimposed ESR spectra which are only well resolved when the temperature is greater than 30 degrees C. These two spectral components are attributed to the dissociated and non-dissociated forms of the fatty acid carboxylic group, anchored at two different positions in the polar interface constituted by the hydrated lipid polar heads. Results on such interactions of other functional groups (spin-labeled fatty ester and fatty alcohol) are also presented.

Binding Sites↗
PubMed1996

New amino-nitroxide spin labels.

Stable free mono- and diradicals containing reactive primary or secondary amino groups in the side-chain have been synthesized by transesterification of amino-substituted esters with paramagnetic alcohols or from spin-labeled acid derivatives and amines. In the second approach the new radical 18 (1-oxyl-3-(2-bromoethoxycarbonyl)-2,2,5,5-tetramethylpyrroline) is proposed as an efficient alkylating species. The nitroxides described are pH-sensitive spin probes and spin labels potentially useful for a diversity of ESR applications in chemistry and biology. New spin-labeled tyramine 16 (N-(1-oxyl-3-carbonyl-2,2,5,5-tetramethyl-pyrroline)tyramine) was successfully employed in a novel assay of protein oxidative damage.

Cyclic N-Oxides↗
PubMed2005

Site-directed spin labeling electron paramagnetic resonance study of the calcium-induced structural transition in the N-domain of human cardiac troponin C complexed with troponin I.

Calcium-induced structural transition in the amino-terminal domain of troponin C (TnC) triggers skeletal and cardiac muscle contraction. The salient feature of this structural transition is the movement of the B and C helices, which is termed the "opening" of the N-domain. This movement exposes a hydrophobic region, allowing interaction with the regulatory domain of troponin I (TnI) as can be seen in the crystal structure of the troponin ternary complex [Takeda, S., Yamashita, A., Maeda, K., and Maeda, Y. (2003) Nature 424, 35-41]. In contrast to skeletal TnC, Ca(2+)-binding site I (an EF-hand motif that consists of an A helix-loop-B helix motif) is inactive in cardiac TnC. The question arising from comparisons with skeletal TnC is how both helices move according to Ca(2+) binding or interact with TnI in cardiac TnC. In this study, we examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a cardiac TnC monomer state and TnC-TnI binary complex by means of site-directed spin labeling electron paramagnetic resonance (EPR). Doubly spin-labeled TnC mutants were prepared, and the spin-spin distances were estimated by analyzing dipolar interactions with the Fourier deconvolution method. An interspin distance of 18.4 A was estimated for mutants spin labeled at G42C on the B helix and C84 on the D helix in a Mg(2+)-saturated monomer state. The interspin distance between Q58C on the C helix and C84 on the D helix was estimated to be 18.3 A under the same conditions. Distance changes were observed by the addition of Ca(2+) ions and the formation of a complex with TnI. Our data indicated that the C helix moved away from the D helix in a distinct Ca(2+)-dependent manner, while the B helix did not. A movement of the B helix by interaction with TnI was observed. Both Ca(2+) and TnI were also shown to be essential for the full opening of the N-domain in cardiac TnC.

Binding Sites↗
PubMed2001

Mechanism of relaxation enhancement of spin labels in membranes by paramagnetic ion salts: dependence on 3d and 4f ions and on the anions.

Progressive saturation EPR measurements and EPR linewidth determinations have been performed on spin-labeled lipids in fluid phospholipid bilayer membranes to elucidate the mechanisms of relaxation enhancement by different paramagnetic ion salts. Such paramagnetic relaxation agents are widely used for structural EPR studies in biological systems, particularly with membranes. Metal ions of the 3d and 4f series were used as their chloride, sulfate, and perchlorate salts. For a given anion, the efficiency of relaxation enhancement is in the order Mn(2+) > or = Cu(2+) > Ni(2+) > Co(2+) approximately Dy(3+). A pronounced dependence of the paramagnetic relaxation enhancement on the anion is found in the order ClO(-)(4) > Cl(-) > SO(2-)(4). This is in the order of the octanol partition coefficients multiplied by spin exchange rate constants that were determined for the different paramagnetic salts in methanol. Detailed studies coupled with theoretical estimates reveal that, for the chlorides and perchlorates of Ni(2+) (and Co(2+)), the relaxation enhancements are dominated by Heisenberg spin exchange interactions with paramagnetic ions dissolved in fluid membranes. The dependence on membrane composition of the relaxation enhancement by intramembrane Heisenberg exchange indicates that the diffusion of the ions within the membrane takes place via water-filled defects. For the corresponding Cu(2+) salts, additional relaxation enhancements arise from dipolar interactions with ions within the membrane. For the case of Mn(2+) salts, static dipolar interactions with paramagnetic ions in the aqueous phase also make a further appreciable contribution to the spin-label relaxation enhancement. On this basis, different paramagnetic agents may be chosen to optimize sensitivity to different structurally correlated interactions. These results therefore will aid further spin-label EPR studies in structural biology.

Anions↗
PubMedDate not supplied

[Study of the irreversible conformation change of immunoglobulin M by spin-labeling at the carbohydrate and peptide moieties of the molecule].

The irreversible conformational change of the immunoglobulin M (IgM) molecule (Waldenström disease) at pH approximately 3 was studied by means of spin-labels introduced in the carbohydrate (2,2,6,6,-tetramethyl-4-aminopiperidine-1-oxyl) and peptide (2,2,5,5,-tetramethyl-3-(dichloro-symm.-triazinylamino)-pyrrolidine-1-oxyl) moieties of the molecule. A marked rise of structure density of IgM especially in the (Fc)5-region and some minor local conformational changes in the Fab-regions were found. Comparison of our findings with the published data shows that Fab-regions of the principal immunoglobulins are rigid structures. Steric hindrance for Fab-regions increases markedly in the row Fab--F(ab')2--IgG--IgA--IgM restricting their spatial mobility. Monomeric Fc-regions of IgM are evidently flexible and one of the domains is especially mobile. It is supposed that oligosaccharide groups of IgM are of two types which differ in their spatial mobility. It was found by ammonium sulfate precipitation of IgM spin-labeled at the peptide moiety that the relative mobility of amino acid residues coupled with spin-label is strongly restricted.

Carbohydrate Conformation↗