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PubMed2000

Membrane location of spin-labeled cytochrome c determined by paramagnetic relaxation agents.

The mitochondrial protein horse heart cytochrome c was specifically spin-labeled with succinimidyl-2,2,5, 5-tetramethyl-3-pyrroline-1-oxyl-carboxylate on different lysine residues at positions 86, 87, 72, 8, or 25, respectively. Site-specifically labeled species were separated chromatographically and identified by peptide sequencing of tryptic digests. The monolabeled protein was bound to negatively charged phospholipid membranes composed of dioleoylphosphatidylglycerol, and the accessibility of the spin-labeled lysine residues to lipid-soluble molecular oxygen and to lipid-impermeant chromium maltolate was determined from the saturation properties of the ESR spectra. The accessibilities of the spin-labeled proteins relative to those obtained for phospholipids spin-labeled in the headgroup region, in the presence of unlabeled protein, identify the position of the spin-labeled lysine residues relative to the phospholipid bilayer surface. We have found that cytochrome c does not penetrate into the membrane interior and that the active side of cytochrome c in the protein-membrane interaction is the side on which lys86, lys87, and lys72 are located.

Animals↗
PubMed1998

Ligand-induced conformational change in the ferric enterobactin receptor FepA as studied by site-directed spin labeling and time-domain ESR.

A mutant of the ferric enterobactin receptor, FepA, containing a valine to cysteine (V338C) substitution was made and the purified protein selectively modified with a sulfhydryl-specific nitroxide spin label. In reconstituted liposomes, interaction of the attached spin label with a combination of water-soluble and lipid-soluble relaxation agents indicated that the V338C site was located in the polar headgroup region of the membrane, approximately 1.5-4.5 A above the phosphate groups of the lipids. Binding of the ligand, ferric enterobactin (FeEnt), to the purified spin-labeled protein produced a significant decrease in both the rotational freedom and accessibility of the nitroxide, indicating the formation of new structural contacts between the spin label and either the protein or the bound ligand. Electron spin-echo (ESE) measurements of the nitroxide phase-memory relaxation rate in the presence and absence of bound ligand showed substantial dipolar coupling between the Fe3+ of FeEnt and the spin label and provided an iron-nitroxide distance estimate in the range of 20-30 A. We conclude that the ligand-induced changes in spin label motion and accessibility are due to new tertiary contacts with the protein and not to direct contact with the ligand. These studies suggest that V338C may occupy a hinge region connecting the ligand binding surface loop to the beta-barrel and provide the strongest evidence to date of an in vitro ligand-induced conformational change in FepA.

Bacterial Outer Membrane Proteins↗
PubMed1980

Proton involvement with the light-induced hindrance of spin label motion in the lumen of spinach thylakoids.

The light-induced hindrance of spin label motion increases linearly with light intensity. However, it has not been possible to unambiguously demonstrate light saturation due to the very high rates of spin label reduction at high light intensity. The light-induced hindrance of spin label motion may be mimicked in the dark by subjecting thylakoids to appropriately low pH regimes. Uncouplers such as gramicidin-D and methylamine reduce the light-induced hindrance to dark levels as does ethylenedinitrilotetraacetate (EDTA) treatment. Valinomycin plus KCl which destroys the electric potential is only partially effective in reducing the light-induced hindrance. These results indicate that protons in the aqueous lumen of the thylakoids are closely involved with the observed light-induced hindrance of spin label motion.

Chloroplasts↗
PubMed1993

An iodoacetamide spin-label selectively labels a cysteine side chain in an occluded site on the sarcoplasmic reticulum Ca(2+)-ATPase.

Sarcoplasmic reticulum vesicles were labeled with [14C]iodoacetamide spin-label (ISL) under conditions where time courses of the reaction predicted that one amino acid residue would be preferentially labeled. Solubilized tryptic peptides were separated by high-performance liquid chromatography following extensive digestion, and amino acid sequences were determined for major and minor radio-labeled peptides. Only one radio-labeled residue, Cys-674 on the Ca(2+)-ATPase, could be identified. Extensive incubation with excess label increased nonspecific labeling, but did not produce detectable amounts of any other reactive side chain residue. Time courses of the iodoacetamide spin-label reaction were compared to those of 6-(iodoacetamido)fluorescein (IAF), and the ISL reaction was found to be more selective, in accordance with previous studies showing that IAF labeled both Cys-674 and Cys-670 [Bishop, J. E., Squire, T. C., Bigelow, D. J., & Inesi, G. (1988) Biochemistry 27, 5233-5240]. Titrations with spin-broadening reagents NiCl2 and Ni-EDTA showed Cys-674 to be in a region with very low solvent accessibility. These titrations also showed the ATPase to be distributed between two alternating conformations based on the accessibility of the label to NiCl2.

Amino Acid Sequence↗
PubMed1991

Platelet membrane fluidity in Alzheimer's disease and multi-infarct dementia: a spin label study.

The membrane fluidity of platelets isolated from 15 patients with probable Alzheimer's disease (AD), 11 patients with probable multi-infarct dementia (MID), and 7 neurologically healthy controls was studied by electron spin resonance (ESR) spectroscopy employing spin label techniques. Spin label I(12,3) probed the shallow site (hydrophilic region) and spin label I(5, 10) the deeper site (hydrophobic region) of the platelet membrane. With both probes, a significant increase in membrane fluidity was observed in patients with AD and MID, as compared to age-matched controls. However, there were no significant differences in fluidity between AD and MID patients. Our results suggest an increased platelet membrane fluidity in dementias, but the change seems not to be specific to AD.

Aged↗
PubMed1976

Spin labeled nucleic acids.

Homopolyribonucleotides and E. coli DNA wer spin labeled with an iodoacetamide-nitroxide compound. The extent of labeling is highly dependent upon the nature of the base and the secondary structure of the nucleic acid. This spin label-polymer linkage is unstable at high temperatures and in phosphate buffers. In order to determine the effect of changes in the environment of nucleic acids on the esr signals of their attached spin labels, the polynucleotides were subjected to temperature and viscosity perturbations. An increase in temperature (T) affects a linear decrease in the anisotropy factor of the esr signal. The log tau (tau = correlation time) versus (1/T) profile is linear with a positive slope when the spin label is attached to single stranded polynucleotides but exhibits discontinuities at certain critical temperatures when attached to the duplexes poly (As-U) and poly (I-Cs). These critical temperatures are lower than the optical Tm. Logarithmic increase in viscosity was found to produce a linear increase in tau in aqueous sucrose solutions.

Binding Sites↗
PubMed1999

Determination by photoreduction of flip-flop kinetics of spin-labeled stearic acids across phospholipid bilayers.

Spin-labeled stearic acid derivatives (N-DS) can be used to determine the rate at which lipid-derived drugs can cross a phospholipid bilayer (flip-flop). The flip-flop rate of N-DS (where N=5, 6, 7, 9, 10, 12, 16), was measured using vectorial photoreduction of nitroxides to their corresponding hydroxylamine by FMN, a charged, membrane-impermeable flavin, by hydrogen atom transfer from EDTA. From the time difference in the photoreduction rates of N-DS located in the outer and inner half of the bilayer, the flip-flop rate of N-DS across the bilayer can be determined. The results show that at pH 8.0 or lower, the photoreduction of 5-DS on one side of the membrane by FMN is slower than the flip-flop rate of 5-DS across phospholipid bilayers. For 5-DS at pH 7.0, this rate is at least 33.8+/-4.24 s or faster. Stearic acids with the spin label at different positions along the acyl chain (N=5, 6, 7, 9, 10, 12) have similar flip-flop rates in the liposomes at pH 7.0 although 16-DS is slower, probably due to the inaccessibility of the nitroxide moiety to FMN. It is most likely that the fast distribution of 5-DS in cells is due to the fast movement of acidic form, but not the salt form, of 5-DS across membrane bilayers. The oxazolidine (nitroxide moiety) does not seem to affect the pKa ( approximately 8.3) of stearic acid at air-water interface. Thus, N-DS are good probes for studying the distribution kinetics of stearic acid derivatives in biological systems.

Cyclic N-Oxides↗
PubMedDate not supplied

[Spectral division of conformational states of spin-labeled tRNA Phe from Escherichia coli].

A spectral division method of two conformational states of spin-labeled macromolecules is presented. The method is suitable in conditions of highly anisotropic motion of spin label and is based on titration of experimental spectra of spin-labeled macromolecule by theoretical ones. Theoretical spectra simulation uses the Freed theory and spin-Hamiltonian parameters, derived from independent experiments. Nomogrammes and formula for calculation of order parameter Sz and correlation time tau c in temperature-viscosity experiment are available. The method was applied to spectral division of two conformational states of spin-labeled tRNAPhe from E. coli and spectral parameters Sz and tau c were obtained for both states. ESR spectra of these conformational states at t degree = 20 degrees differ strongly from one another by order parameter Sz. The first conformer, that is characterised by a greater order parameter has no globular conformational transition (in terms of changes of the hydrodynamic macromolecule radius) between 2 degrees and 20 degrees, but local conformational changes take place in this temperature region.

Electron Spin Resonance Spectroscopy↗
PubMedDate not supplied

Exploring the local conformational space of a membrane protein by site-directed spin labeling.

Molecular modeling based on a hybrid evolutionary optimization and an information condensation algorithm, called GHOST, of spin label ESR spectra was applied to study the structure and dynamics of membrane proteins. The new method is capable of providing detailed molecular information about the conformational space of the spin-labeled segment of the protein in a membrane system. The method is applied to spin-labeled bacteriophage M13 major coat protein, which is used as a model membrane protein. Single cysteine mutants of the coat protein were labeled with nitroxide spin labels and incorporated in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers. The new computational method allows us to monitor distributions of local spatial constraints and molecular mobility, in addition to information about the location of the protein in a membrane. Furthermore, the results suggest that different local conformations may coexist in the membrane protein. The knowledge of different local conformations may help us to better understand the function-structure relationship of membrane proteins.

Algorithms↗
PubMed1986

Two-dimensional 1H NMR of three spin-labeled derivatives of bovine pancreatic trypsin inhibitor.

Three nitroxide spin-labeled monoderivatives of bovine pancreatic trypsin inhibitor were prepared with the amino-specific reagent succinimidyl 1-oxy-2,2,5,5-tetramethyl-3-pyrroline-3-carboxylate. The monoderivatives were purified by ion-exchange and affinity chromatography. Thin-layer maps of tryptic peptides of the monoderivatives showed that the spin-label was incorporated at either the alpha-amino group, Lys-15, or Lys-26. Two-dimensional J-correlated 1H NMR spectra of the monoderivatives were recorded. Spectra were also recorded after reduction by ascorbic acid of the nitroxide label to hydroxylamine. With the nitroxide label present, significant line-broadening effects on many of the cross peaks in the spectra were observed. The extent of line broadening for the C alpha H-NH cross peaks was qualitatively correlated with the distance between the labeled amino group and the average C alpha H-NH position in the crystal structure. The spin-label affects cross peaks of protons within approximately 15 A. This study suggests that it is feasible to accumulate sufficient intramolecular distances in order to determine protein solution structures with the aid of distance geometry algorithms.

Amino Acids↗
PubMed2001

Interaction of cholesterol with sphingomyelin in mixed membranes containing phosphatidylcholine, studied by spin-label ESR and IR spectroscopies. A possible stabilization of gel-phase sphingolipid domains by cholesterol.

The ESR spectra from different positional isomers of sphingomyelin and phosphatidylcholine spin-labeled in their acyl chain have been studied in sphingomyelin(cerebroside)-phosphatidylcholine mixed membranes that contain cholesterol. The aim was to investigate mechanisms by which cholesterol could stabilize possible domain formation in sphingolipid-glycerolipid membranes. The outer hyperfine splittings in the ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled on the 5 C atom of the acyl chain were consistent with mixing of the components, but the perturbations on adding cholesterol were greater in the membranes containing sphingomyelin than in those containing phosphatidylcholine. Infrared spectra of the amide I band of egg sphingomyelin were shifted and broadened in the presence of cholesterol to a greater extent than the carbonyl band of phosphatidylcholine, which was affected very little by cholesterol. Two-component ESR spectra were observed from lipids spin-labeled on the 14 C atom of the acyl chain in cholesterol-containing membranes composed of sphingolipids, with or without glycerolipids (sphingomyelin/cerebroside and sphingomyelin/cerebroside/phosphatidylcholine mixtures). These results indicate the existence of gel-phase domains in otherwise liquid-ordered membranes that contain cholesterol. In the gel phase of egg sphingomyelin, the outer hyperfine splittings of sphingomyelin spin-labeled on the 14-C atom of the acyl chain are smaller than those for the corresponding spin-labeled phosphatidylcholine. In the presence of cholesterol, this situation is reversed; the outer splitting of 14-C spin-labeled sphingomyelin is then greater than that of 14-C spin-labeled phosphatidylcholine. This result provides some support for the suggestion that transbilayer interdigitation induced by cholesterol stabilizes the coexistence of gel-phase and "liquid-ordered" domains in membranes containing sphingolipids.

Brain Chemistry↗
PubMed1977

Effects of spin labeled acetylcholine analogs on cholinergic receptors of the leech.

The spin labeled acetylcholine analog, SL-2, which is a potent, non-stimulating muscarinic antagonist on the isolated frog heart, is a potent agonist a low concentrations (3x10-5 M) and an antagonist at slightly higher concentrations (4.5x10-5 M) on the mixed cholinergic receptors of the leech. Ion the other hand, the spin labeled acetylcholine analog, SL-1, possesses no agonist activity while it retains the antagonist activity of SL-2. Furthermore, the spin labeled analog, SL-3, which causes hyperpolarization and a decrease in action potential frequency, exhibits neither agonist nor antagonist activity.

Acetylcholine↗
PubMed1985

Strong interactions between a spin-labeled cholesterol analog and erythrocyte proteins in the human erythrocyte membrane.

We have used a spin label analog of cholesterol bearing a nitroxide on the alkyl chain (26-nor-25-doxylcholestanol) to study cholesterol-protein interactions in the human erythrocyte membrane. As judged from the ESR spectrum, the spin label is readily incorporated into the membrane when added from a concentrated ethanolic solution to a cell or ghost suspension. With intact erythrocytes or white ghosts in isotonic buffer, the ESR spectrum is a superposition of a mobile component and a strongly immobilized component (outer hyperfine splitting 61-63 G). The latter corresponds to approx. 45% of the signal, a percentage which is barely affected by varying the temperature between 5 and 37 degrees C. Removal of the cytoskeletal proteins spectrin and actin by low ionic strength treatment or of all extrinsic proteins by alkali treatment of ghosts reduces the immobilized fraction to approx. 25%. The effect of controlled proteolysis of intrinsic proteins was also tested. Pre-treatment of cells with chymotrypsin or pre-treatment of unsealed ghosts with trypsin has no effect on the ESR spectrum obtained with alkali-treated membranes. On the other hand, after chymotrypsin treatment of unsealed ghost, which reduces the band 3 protein to a 17.5 kDa membrane fragment, the strongly immobilized component is no longer observable. These data show that the cholesterol analog 26-nor-25-doxylcholestanol interacts strongly with one or several proteins of the erythrocyte membrane. That the intrinsic protein band 3 is involved is suggested by the disappearance of the immobilized fraction occurring upon chymotrypsin digestion of this protein. Our results are thus consistent with the proposal of a selective cholesterol-band 3 interaction in the erythrocyte membrane (Schubert, D. and Boss, K. (1982) FEBS Lett. 150, 4-8). Our data also suggest that this interaction is influenced by cytoskeletal proteins, an effect which can be explained considering the known linking of band 3 to the erythrocyte cytoskeleton via ankyrin. Experiments have also been carried out with 3-doxylandrostanol, a more commonly used cholesterol spin-label analog. With this spin label, at all temperatures investigated, we found it impossible to demonstrate unambiguously the existence of two spectral components. It is suggested that 26-nor-25-doxylcholestanol is a better reporter of cholesterol behavior in membranes.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1983

A new spin label method for the measurement of erythrocyte internal microviscosity.

A new spin-label method for the measurement of the internal microviscosity of erythrocyte is presented. The spin label used is 2,2',5,5'-tetramethyl-3-maleimidopyrrolidinyl-N-oxyl (MAL-5) which penetrates inside the red blood cell and binds covalently on cytoplasmic glutathione. After washing off the external label, 98% of the electron paramagnetic signal is due to the labelled glutathione. This signal allows one to measure the rotational correlation time of the label. A calibration curve established with spin-labelled glutathione in sucrose solutions of increasing viscosity is used to convert the measured rotation times into viscosity units. This method avoids the use of unphysiological salts like potassium ferricyanide, and permits the study of red blood cells in various suspension media. In normal human subjects, the mean value of microviscosity is 4.45 +/- 0.16 mPa . s at 20 degrees C in isotonic saline (25 subjects) and 6 +/- 0.25 mPa . s in plasma. The variations of microviscosity as a function of the osmolarity of the medium are explained according to a theoretical model taking into account the variations of the red blood cell volume and the viscometric properties of haemoglobin.

Adolescent↗
PubMed1980

Orientation of spin-labeled myosin heads in glycerinated muscle fibers.

We have used electron paramagnetic resonance (EPR) spectra to study spin labels selectively and rigidly attached to myosin heads in glycerinated rabbit psoas muscle fibers. Because the angle between the magnetic field and the principal axis of the probe determines the position of the EPR absorption line, spectra from labeled fibers oriented parallel to the magnetic field yielded directly the distribution of spin label orientations relative to the fiber axis. Two spin labels, having reactivities resembling iodoacetamide (IASL) and maleimide (MSL), were used. In rigor fibers with complete filament overlap, both labels displayed a narrow angular distribution, full width at half maximum approximately 15 degrees, centered at angles of 68 degrees (IASL) and 82 degrees (MSL). Myosin subfragments (heavy meromyosin and subfragment-1) were labeled and allowed to diffuse into fibers. The resulting spectra showed the same sharp angular distribution that was found for the labeled fibers. Thus is appears that virtually all myosin heads in a rigor fiber have the same orientation relative to the fiber axis, and this orientation is determined by the actomyosin bond. Experiments with stretched fibers indicated that the spin labels on the fraction of heads not interacting with actin filaments had a broad angular distribution. Addition of ATP to unstretched fibers under relaxing conditions produced orientational disorder, resulting in a spectrum almost indistinguishable from that of an isotropic distribution of probes. Addition of either an ATP analog (AMPPNP) or pyrophosphate produced partial disorder. That is a fraction of the probes remained sharply oriented as in rigor while a second fraction was in a disordered distribution similar to that of relaxed fibers.

Actins↗
PubMedDate not supplied

[Differentiation of the spatial mobility of lysine residues in immunoglobulin G using spin labels].

Some dynamic and conformational changes of Lys residue in molecules of human IgG have been studied by the spin label method using N-(1-oxyl-2,2,5,5-tetramethyl-3- pyrrolidinyl)maleimide, known to selectively modify the Lys residue in human IgG. The value of tau 20 C of the spin labelled IgG was found to be 26 +/- 2 ns, what is known to be characteristic for the labelling of the peptide residue in IgG. It has also been found that the spin-labelled Lys residues exist in two forms with different mobility and rigidity with the ratio of mobile to rigid forms of 3:1. The hypothesis that the reason for immobility of Lys residues in IgG lies in their drawing in the H-bonds formation is being proved. The study by the spin label method has shown that the often observed mobilization of the EPR spectra of the spin labelled IgG is caused by the process of autolysis in the stored samples. The use of the phenylmethylsulfonylfluoride can completely eliminate this phenomena.

Electron Spin Resonance Spectroscopy↗
PubMed1978

Electron spin resonance and nuclear relaxation studies on spin-labeled glutamate dehydrogenase.

The reaction of glutamate dehydrogenase with two different stable nitroxides (spin labels) is reported. The two compounds contain a carbonyl and an iodoacetamide group as their reactive parts. The carbonyl compound inactivates the enzyme by the formation of a 1:1 covalent complex after NaBH4 reduction of an intermediate Schiff's base. Evidence indicates that the enzyme is modified at lysine-126 in the active site. The electron spin resonance (ESR) spectrum of spin-labeled enzyme indicates a high degree of immobilization of the nitroxide. The binding of reduced coenzyme NADPH is reflected by a change (immobilization) of the ESR spectrum. Nuclear relaxation of bound substrate, oxidized coenzyme, and inhibitor by the paramagnetic group is observed. This shows the existence of a binding site for these compounds close to the active site. The distances of selected protons of the binding ligands to the nitroxide are calculated. The iodoacetamide spin label reacts with several groups, one of which is not a sulfhydryl. The reaction of this particular group causes inactivation of the enzyme. Protection against this inactivation could be achieved with certain ligands. Only enzyme that was spin labeled without such protection caused paramagnetic relaxation of bound substrate and coenzyme.

Allosteric Site↗
PubMed2003

A spin labelling study of immunomodulating peptidoglycan monomer and adamantyltripeptides entrapped into liposomes.

The interaction of immunostimulating compounds, the peptidoglycan monomer (PGM) and structurally related adamantyltripeptides (AdTP1 and AdTP2), respectively, with phospholipids in liposomal bilayers were investigated by electron paramagnetic resonance spectroscopy. (1). The fatty acids bearing the nitroxide spin label at different positions along the acyl chain were used to investigate the interaction of tested compounds with negatively charged multilamellar liposomes. Electron spin resonance (ESR) spectra were studied at 290 and 310 K. The entrapment of the adamantyltripeptides affected the motional properties of all spin labelled lipids, while the entrapment of PGM had no effect. (2). Spin labelled PGM was prepared and the novel compound bearing the spin label attached via the amino group of diaminopimelic acid was chromatographically purified and chemically characterized. The rotational correlation time of the spin labelled molecule dissolved in buffer at pH 7.4 was studied as a function of temperature. The conformational change was observed above 300 K. The same effect was observed with the spin labelled PGM incorporated into liposomes. Such effect was not observed when the spin labelled PGM was studied at alkaline pH, probably due to the hydrolysis of PGM molecule. The study of possible interaction with liposomal membrane is relevant to the use of tested compounds incorporated into liposomes, as adjuvants in vivo.

Acetylmuramyl-Alanyl-Isoglutamine↗