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PubMed1978

Tertiary structure variability within the quaternary states of hemoglobin: a spin label study.

Using variable temperature techniques, the spin label spectral resolution of hemoglobin labeled at the beta93 cysteines with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodonacetamide has been greatly enhanced. The effects of different ligands, inositol hexaphosphate, pH and salt concentration upon spin labeled ferrous and ferric hemoglobin indicate that the beta chain tertiary structure exhibits considerable variability within the oxy and deoxy quaternary structures. From these studies ligand and spin state changes both appear to be of significance in producing structural changes; binding of inositol hexaphosphate then produces further structural changes secondary in amplitude.

Chemical Phenomena↗
PubMed1992

Orientational distribution of spin-labeled actin oriented by flow.

Previous studies on spin-labeled F-actin (MSL-actin), using saturation transfer electron paramagnetic resonance (ST-EPR), have demonstrated that actin has submillisecond rotational flexibility and that this flexibility is affected by the binding of myosin and its subfragments. This rotational flexibility does not change during the active interaction of myosin heads, actin, and adenosine triphosphate. However, these ST-EPR studies, performed on randomly oriented actin, would not be sensitive to orientational changes on the millisecond time scale or slower. In the present study, we have clarified these results by performing conventional EPR experiments on MSL-actin oriented by flow to detect changes in the orientational distribution. We have determined the orientational distribution of the spin labels relative to the magnetic field (flow direction) by comparing experimental EPR spectra to simulated EPR spectra corresponding to known orientational distributions. Spectra acquired during flow indicate two populations of probes: a highly ordered population and a disordered population. For the ordered population (28% of the total spin concentration), the angle between the actin filament axis and the nitroxide z axis (theta) fits a Gaussian distribution centered at 32.0 +/- 0.9 degrees, with a full width at half maximum of 20.7 +/- 3.9 degrees. The angle between the nitroxide x axis and the projection of the field in the xy plane (phi) is centered at 37.5 +/- 9.2 degrees with a full width of 24.9 +/- 10.7 degrees. This orientational distribution is not significantly changed upon the binding of phalloidin or myosin subfragment 1 (S1), indicating that these proteins do not affect the axial orientation of actin subunits. Spectra of spin-labeled S1 (MSL-S1) bound to actin oriented by flow have about the same orientational distribution as MSL-S1 bound to actin in oriented fibers. Thus, the oriented fraction of flow-oriented actin filaments has nearly the same high degree of alignment as the actin filaments in muscle fibers.

Actins↗
PubMed2005

Conformational change in full-length mouse prion: a site-directed spin-labeling study.

The structure of the mouse prion (moPrP) was studied using site-directed spin-labeling electron spin resonance (SDSL-ESR). Since a previous NMR study by Hornemanna et al., [Hornemanna, Korthb, Oeschb, Rieka, Widera, Wüthricha, Glockshubera, Recombinant full-length murine prion protein, mPrP (23-231): purification and spectroscopic characterization, FEBS Lett. 413 (1997) 277-281] has indicated that N96, D143, and T189 in moPrP are localized in a Cu(2+) binding region, Helix1 and Helix2, respectively, three recombinant moPrP mutations (N96C, D143C, and T189C) were expressed in an Escherichia coli system, and then refolded by dialysis under low pH and purified by reverse-phase HPLC. By using the preparation, we succeeded in preserving a target cystein residue without alteration of the alpha-helix structure of moPrP and were able to apply SDSL-ESR with a methane thiosulfonate spin label to the full-length prion protein. The rotational correlation times (tau) of 1.1, 3.3, and 4.8ns were evaluated from the X-band ESR spectra at pH 7.4 and 20 degrees C for N96R1, D143R1, and T189R1, respectively. tau reflects the fact that the Cu(2+) binding region is more flexible than Helix1 or Helix2. ESR spectra recorded at various temperatures revealed two phases together with a transition point at around 20 degrees C in D143R1 and T189R1, but not in N96R1. With the variation of pH from 4.0 to 7.8, ESR spectra of T189R1 at 20 degrees C showed a gradual increase of tau from 2.9 to 4.8ns. On the other hand, the pH-dependent conformational changes in N96R1 and D143R1 were negligible. These results indicated that T189 located in Helix2 possessed a structure sensitive to physiological pH changes; simultaneously, N96 in the Cu(2+) binding region and D143 in Helix1 were conserved.

Animals↗
PubMed1988

Interactions of spin-labeled calmodulin with trifluoperazine and phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III).

Bovine calmodulin analogues, spin-labeled at methionine and tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies designed to investigate calmodulin interactions with the antipsychotic drug trifluoperazine and the calmodulin-binding protein 3',5'-cyclic nucleotide phosphodiesterase. Trifluoperazine titrations of spin-labeled calmodulin analogues were carried out in the presence of Ca(II), Cd(II), and Tb(III). Similar experiments were performed with the phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III). EPR signals from the methionine-directed probe proved to be more sensitive to the binding of target molecules than signals from the tyrosine-directed probe, perhaps indicating that the spin-labeled methionine is at a site close to the target molecule binding site. While the binding of TFP, as monitored by EPR spectral changes in the methionine spin-labeled calmodulin, was in evidence with Ca(II), Cd(II), and all the lanthanides examined, no binding of phosphodiesterase to calmodulin could be detected in the presence of the lanthanide ions, perhaps due to inactivation of the phosphodiesterase by lanthanide ion binding. The abilities of the spin-labeled calmodulins to activate phosphodiesterase were also investigated. The spin-labeled tyrosine calmodulin was able to activate phosphodiesterase as well as native calmodulin, while a lower degree of activation was found when the spin-labeled methionine analogue was used.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2002

High-field electron spin resonance of spin labels in membranes.

High-field electron spin resonance (ESR) spectroscopy is currently undergoing rapid development. This considerably increases the versatility of spin labelling which, at conventional field strengths, is already well established as a powerful physical technique in membrane biology. Among the unique advantages offered by high-field spectroscopy, particularly for spin-labelled lipids, are sensitivity to non-axial rotation and lateral ordering, a better orientational selection, an extended application to rotational dynamics, and an enhanced sensitivity to environmental polarity. These areas are treated in some depth, along with a detailed consideration of recent developments in the investigation of transmembrane polarity profiles.

Animals↗
PubMed1988

Line-shape analysis of NMR difference spectra of an anti-spin-label antibody.

Specifically deuteriated Fab fragments of the anti-spin-label antibody AN02 were prepared. NMR difference spectra were obtained, in which the spectrum of Fab with some fraction of the binding sites occupied with spin-label hapten was subtracted from the spectrum of Fab with no spin-label. The peak heights were analyzed as a function of the fractional occupation of the binding site, using a computer program that calculates a best fit to the observed spectra. This method treats all of the peaks in the spectra simultaneously. Analyzing all peaks at once allows for the interdependencies in the spectra arising from overlap of positive and negative signals from different peaks. The fitting program calculates line widths for the peaks arising from protons in the binding site region. Almost all of the line widths calculated for the spectrum of the Fab complex with diamagnetic hapten dinitrophenyldiglycine were found to be narrower than the line widths of the corresponding resonances in the spectrum of Fab with an empty binding site. The distances of the binding site region protons from the unpaired electron of the hapten were also obtained from this calculation. Two tyrosine protons were found to be close (less than A) to this electron. These line-width and distance results are discussed with respect to the structure and dynamics of the antibody binding site.

Immunoglobulin Fab Fragments↗
PubMed1978

Reverse transcriptase: a monitor for perturbation effects of spin labels covalently bound to nucleic acids.

The sensitive biological assay for reverse transcriptase was used to monitor potential perturbation effects of spin labels covalently bound to various nucleic acids or nucleic acid analogs to the extent of about one label per 100 residues. The inhibitory properties of the spin labeled and unlabeled biopolymers were compared for evaluating possible interference of the reporter group in protein-nucleic acid interaction studies. The amount of inhibitor required for 50% inhibition (ED50) was determined for the competitive inhibitors (U)n, l(U)n, (RUGT,U)n, (Um)n, (A)n, (Am)n, and l(A)n as well as for thenon- or uncompetitive inhibitors (dUfl)n, l(dUfl)n, (dUz)n, and l(dUz)n. The most pronounced inhibition was observed with spin labeled and unlabeled (dUfl)n. The results indicate that the ED50 and the kinetic patterns of inhibition are similar for the spin labeled and unlabeled inhibitors studied. Thus, the presence of a limited number of spin labels in a nucleic acid matrix has little effect, if any, on reverse transcriptase-nucleic acid complexes and most likely on other protein-nucleic acid complexes.

Avian Myeloblastosis Virus↗
PubMed1999

Membrane assembly of the 16-kDa proteolipid channel from Nephrops norvegicus studied by relaxation enhancements in spin-label ESR.

The 16-kDa proteolipid from the hepatopancreas of Nephrops norvegicus belongs to the class of channel proteins that includes the proton-translocation subunit of the vacuolar ATPases. The membranous 16-kDa protein from Nephrops was covalently spin-labeled on the unique cysteine Cys54, with a nitroxyl maleimide, or on the functionally essential glutamate Glu140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). The intensities of the saturation transfer ESR spectra are a sensitive indicator of spin-spin interactions that were used to probe the intramembranous structure and assembly of the spin-labeled 16-kDa protein. Spin-lattice relaxation enhancements by aqueous Ni(2+) ions revealed that the spin label on Glu140 is located deeper within the membrane (around C9-C10 of the lipid chains) than is that on Cys54 (located around C5-C6). In double labeling experiments, alleviation of saturation by spin-spin interactions with spin-labeled lipids indicates that spin labels both on Cys54 and on Glu140 are at least partially exposed to the lipid chains. The decrease in saturation transfer ESR intensity observed with increasing spin-labeling level is evidence of oligomeric assembly of the 16-kDa monomers and is consistent with a protein hexamer. These results determine the locations and orientations of transmembrane segments 2 and 4 of the 16-kDa putative 4-helix bundle and put constraints on molecular models for the hexameric assembly in the membrane. In particular, the crucial DCCD-binding site that is essential for proton translocation appears to contact lipid.

Animals↗
PubMed2001

A spin-labeled abasic DNA substrate for AP endonuclease.

We report the first observation of a spin-labeled ds 23-mer oligonucleotide by high-field electron spin resonance (ESR) and demonstrate that it interacts with AP endonuclease, the key enzyme in DNA abasic site repair. The spin labeled 23-mer with a U at position 12 of the upper strand is processed by uracil DNA glycosylase to provide the abasic substrate. With a spin-label two nucleotides away from the abasic site, AP endo binds and cleaves when the label is 3' but not 5' to the abasic site. These results confirm that the disposition of the bases immediately upstream of the abasic site is particularly critical for cleavage by AP endo, and establish that DNA-protein interactions in this important enzyme can be examined using spin-labeled substrates.

Carbon-Oxygen Lyases↗
PubMed2001

Analysis of electron spin resonance spectra of alkyl spin labels in excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes.

The electron spin resonance (ESR) spectra of alkyl spin labels were observed in the excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes. The spectrum of 5-doxylstearic acid (5-NS) in the stratum corneum and order parameter obtained from the spectrum, indicated that the spin label was present in highly ordered lipid lamella. On the other hand, the spectrum of methyl ester of 5-NS (5-NMS) and its apparent rotational correlation time calculated from the spectrum, showed only a weakly immobilized component in the stratum corneum as well as in the whole excised skin. The ester spin label seemed to be scarcely present in the rigid lipid lamella, but mainly in the relatively fluid environment. On the other hand, cationic alkyl spin labels showed quite different spectra depending on their alkyl chain lengths. Long-chain 4-(N,N-dimethyl-N,-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-15) seemed to be present in the protein region of the stratum corneum as we recently reported, whereas hydrophilic quaternary ammonium spin label 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-1) seemed to be present in the bulk water of the skin, even in delipidized skin. These findings indicated that the different interaction and different localization of the alkyl spin labels depended on their electronic charge as well as their alkyl chain lengths.

Animals↗
PubMed1984

Studies on the property of sulfhydryl binding site on the lung normal and cancer cell membrane of Chinese hamster with maleimide spin labels.

In this paper, five maleimide spin labels with different chain lengths were used to study the properties of binding sites of sulfhydryl groups on the membrane proteins of normal cell V79 and cancer cells V79-B1 in the lung of Chinese hamster. The ratio of the strongly immobilized component to the weakly immobilized component (s/w) and the rotational correlation time (tau c) were calculated on the basis of ESR spectra. From the varying of the s/w and tau c with the chain lengths of spin labels it was deducted that the binding sites of sulfhydryl groups on V79 and V79-B1 membrane proteins were of conical shape and that the binding sites of sulfhydryl groups on V79 membrane protein were deeper and narrower than those on V79-B1.

Animals↗
PubMed1987

Spin-labeled antitumor derivatives of podophyllotoxin.

Three spin-labeled antitumor derivatives of podophyllotoxin, containing an iminoxyl radical, have been synthesized. Preliminary pharmacological tests showed that these derivatives have significant antitumor activity in several experimental tumor systems with a marked decrease in toxicity compared with the parent compound. The relative intensities of ESR signals of these spin-labeled compounds were linearly proportional to their concentrations, the levels of the agent in blood and organs of mice or rabbits can be measured via ESR spectrometry.

Animals↗
PubMedDate not supplied

Synthesis and application of novel bifunctional spin labels.

The synthesis of new bifunctional spin-labeled cross-linking reagents is described. Covalent attachment to papain was achieved via a thiol-specific thiosulfonate residue and, for the second anchor point, via a nonspecific photoreactive azido function. The thiosulfonate formed a reversible disulfide linkage, which could be cleaved again reductively by dithiothreitol. The spin label, a pyrroline-1-oxyl radical, was highly immobilized after attachment to papain by both functional groups and showed little if any relative motion with respect to the protein.

Cross-Linking Reagents↗
PubMed1986

A proton relaxation enhancement investigation of the binding of fatty acid spin labels to human serum albumin.

Proton relaxation enhancement (PRE) values for fatty acid spin labels bound to human serum albumin have been investigated using the inversion-recovery method at 24 MHz. At 0.1 mM protein concentration and a label-to-protein ratio of one-to-one, the PRE value for 12-Doxylsterate-albumin complex is 7.8 +/- 2.3, whereas the PRE values for 5-Doxylstearate and 16-Doxylstearate-albumin complexes are 1.5 +/- 0.6 and 1.7 +/- 0.7, respectively. Addition of 10-fold excess of stearic acid reduced the PRE values nearly to 1, indicating that the strong enhancements arise from direct binding of fatty acid spin labels to human serum albumin. PRE values for all three labels exhibit maxima as a function of the label-to-protein ratio, suggesting multiple binding sites for fatty acid spin labels with labels in the tightest binding sites not resulting in the most effective relaxation. Based on the rates of reduction of ESR signal amplitudes by sodium ascorbate, the difference in PRE values for the three fatty acid spin labels bound to albumin is attributed to the difference in water accessibility of the nitroxide moieties at various positions along the acyl chain, being greater at the C-12 position than at C-5 or C-16 position. The PRE value of 8 for 12-Doxylstearate bound to human serum albumin indicates that this complex may be a suitable paramagnetic contrast agent for in vivo NMR imaging.

Binding Sites↗
PubMed1992

Saturation transfer, continuous wave saturation, and saturation recovery electron spin resonance studies of chain-spin labeled phosphatidylcholines in the low temperature phases of dipalmitoyl phosphatidylcholine bilayers. Effects of rotational dynamics and spin-spin interactions.

The saturation transfer electron spin resonance (STESR) spectra of 10 different positional isomers of phosphatidylcholine spin-labeled in the sn-2 chain have been investigated in the low temperature phases of dipalmitoyl phosphatidylcholine (DPPC) bilayers. The results of continuous wave saturation and of saturation recovery measurements on the conventional ESR spectra were used to define the saturation properties necessary for interpreting the STESR results in terms of the chain dynamics. Spin labels with the nitroxide group located in the center of the chain tended to segregate preferentially from the DPPC host lipids in the more ordered phases, causing spin-spin interactions which produced spectral broadening and had a very pronounced effect on the saturation characteristics of the labels. This was accompanied by a large decrease in the STESR spectral intensities and diagnostic line height ratios relative to those of spin labels that exhibited a higher degree of saturation at the same microwave power. The temperature dependence of the STESR spectra of the different spin label isomers revealed a sharp increase in the rate of rotation about the long axis of the lipid chains at approximately 25 degrees C, correlating with the pretransition of gel phase DPPC bilayers, and a progressive increase in the segmental motion towards the terminal methyl end of the chains in all phases. Prolonged incubation at low temperatures led to an increase in the diagnostic STESR line height ratios in all regions of the spectrum, reflecting the decrease in chain mobility accompanying formation of the subgel phase. Continuous recording of the central diagnostic peak height of the STESR spectra while scanning the temperature revealed a discontinuity at approximately 14-17 degrees C, corresponding to the DPPC subtransition which occurred only on the initial upward temperature scan, in addition to the discontinuity at 29-31 degrees C corresponding to the pretransition which displayed hysteresis on the downward temperature scan.

1,2-Dipalmitoylphosphatidylcholine↗
PubMedDate not supplied

An electron spin resonance spectral study of the dynamics of spin-labelled fatty acids bound to oxidoreductases.

Electron spin resonance (ESR) spectra have been recorded for 5-doxylstearate (I) and 16-doxylstearate (II) in the presence of bacterial luciferase and soybean lipoxygenase. The acids are inhibitors of the enzymes (Kd approximately 2 x 10(-5) M for II bound to luciferase). Using theoretical computer simulations of the ESR line shapes, an effective correlation time of tau eff approximately 3.7 x 10(-9) +/- 0.5 x 10(-9) s is found for the motion of spin label I bound to luciferase. Concentration-dependent sedimentation velocity experiments indicate luciferase is anisotropic and, assuming a prolate ellipsoid of axial ratio less than or equal to 9, a correlation time of tau a approximately 7.7 x 10(-9) s is predicted for rotation about the luciferase long axis. The tightly bound spin-labelled inhibitors, therefore, are proposed to give rise to ESR spectra chiefly reflecting luciferase long axis rotation. The ESR spectra of I and II, bound to lipoxygenase and to luciferase, appear similar.

Chemical Phenomena↗
PubMedDate not supplied

[Dynamic mobility of the histidine-containing domain of spin-labeled lysozyme].

The hen egg-white lysozyme was modified by the spin label (2,2,6,6-tetramethylpiperidine-N1-oxyl-4-iodacetamide) at the single histidine residue His-15. The rotational correlation time of the molecular carrier was found to be defined by the mobility of the histidine-bearing domain and not influenced by the protein monomer shape at pH 4.7 and dimer shape at pH 7.1. The dependence of viscosity at 1 degree C on the distance between outer wide peaks in the immobilized EPR spectra enabled us to evaluate rotational correlation time of the domain. The molecular mass of the latter was close to the data obtained by X-ray analysis. The spin label was highly mobile at room temperature, as the EPR spectrum displayed the triple shape; at 1 degree C it was immobilized. The new general approach to the EPR spectra simulation was applied to all experimental EPR spectra. This approach is based on a substitution of an undefined stochastic process of the spin label reorientation relative to the lysozyme domain by the defined modelled stochastic processes: axial rotation of the nitroxide relative to the preferable axis and angular oscillations of the nitroxide relative to axes of the molecular coordinate system. Each of the modelled stochastic processes leads to a relative partial averaging of the magnetic tensor components. A set of discrete partially averaged states is introduced with the relative cluster of the spin-labelled molecules. The resulting EPR spectrum is assumed to be the sum of EPR spectra from all the clusters. A good fitting of all simulated EPR spectra is obtained.

Animals↗
PubMed1978

Spin-label and deuterium order parameter discrepancies in bilayers: one possible explanation.

We have simulated electron spin resonance spectra of anisotropically immobilized spin labels of the type seen in lipid and soap-like bilayers using a rigorous formalism which explicitly includes the effects of spin-label motion. In most bilayer systems, spin-label experiments have shown lower order parameters then deuterium-label experiments. In the past this apparent decrease in the order parameters was thought to reflect the distortion of the bilayer by the doxyl ring of the spin probes. We wish to report that this type of discrepancy may be due to the neglect of important motional effects in the time-independent effective Hamiltonian formalisms used in previous interpretations of anisotropically immobilized spin label spectra. That the true order parameters may be the same can be shown by including slow motional corrections in the effective Hamiltonian formalism. The larger volume of the doxyl ring may change the apparent order parameter by increasing the importance of the slow motional effects, as opposed to causing a real decrease in the order parameter, as previously proposed.

Chemical Phenomena↗