Biomedical subjects

K Hideg

Publications and source records attributed to K Hideg.

At least 37 records · Page 2Linked to original sources

PubMed1997

Direct evidence for in vivo nitroxide free radical production from a new antiarrhythmic drug by EPR spectroscopy.

The new Class I anti-arrhythmic agent 2,2,5,5-tetramethyl-3-pyrroline-1-carboxamide derivative, is currently being evaluated in clinical trials in patients with a high risk for cardiac arrhythmias. In this study we show that this antiarrhythmic drug can be chemically converted to the nitroxide free radical analog. Further, using an in vivo Electron Paramagnetic Resonance (EPR) spectroscopy model by detecting free radicals in the distal portion of the tail of an anesthetized mouse, we demonstrate that the drug is oxidized to the corresponding nitroxide. In vitro studies using Chinese hamster V79 cells suggest that the oxidation products of the drug, namely, the hydroxylamine and the nitroxide protect against oxidative damage induced by hydrogen peroxide (H2O2). Taken together, our results suggest that, in addition to the antiarrhythmic effects of the parent drug, sufficient levels of nitroxides may accumulate from the parent drug in vivo to provide antioxidant defense to cardiac tissue that may be subject to ischemia and oxidation-driven injury.

Animals↗

PubMed1996

Organization of diphtheria toxin T domain in bilayers: a site-directed spin labeling study.

The diphtheria toxin transmembrane (T) domain was spin-labeled at consecutive residues in a helical segment, TH9. After binding of the T domain to membranes at low pH, the nitroxide side chains generated by spin labeling were measured with respect to their frequency of collision with polar and nonpolar reagents. The data showed that the helical structure of TH9 in solution is conserved, with one face exposed to water and the other to the hydrophobic interior of the bilayer. Measurement of the depth of the nitroxide side chains from the membrane surfaces revealed an incremental change of about 5 angstroms per turn, which is consistent with a transmembrane orientation of an alpha helix. These results indicate that the helix forms the lining of a transmembrane water-filled channel.

Amino Acid Sequence↗

PubMed1996

Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics.

Thirty single cysteine substitution mutants of T4 lysozyme have been prepared and spin-labeled with a sulfhydryl-specific nitroxide reagent in order to systematically investigate the relationship between nitroxide side-chain mobility and protein structure. The perturbation caused by replacement of a native residue with a nitroxide amino acid was assessed from the resulting changes in biological activity, circular dichroism, and free energy of folding. The nitroxide produced context-dependent changes in stability and activity similar to those observed for substitution with natural amino acids at the same site but had little effect on the circular dichroism spectra. At solvent-exposed sites, the structural perturbation appears to be small at the level of the backbone fold. Nitroxide side-chain mobility faithfully reflects the protein tertiary fold at all sites investigated. The primary determinants of nitroxide side-chain mobility are tertiary interactions and backbone dynamics. Tertiary interactions constrain the side-chain mobility to an extent closely correlated with the degree of interaction. At interhelical loop sites, the side chains have a high mobility, consistent with high crystallographic thermal factors. On the exposed surfaces of alpha-helices, the side-chain mobility is not restricted by interactions with nearest neighbor side chains but appears to be determined by backbone dynamics. An unexpected result is a striking difference between the mobility of residues near the C- and N-termini of helices. These results provide the foundation for another dimension of information in site-directed spin-labeling experiments that can be interpreted in terms of the protein tertiary fold, its equilibrium dynamics and time-dependent conformational changes.

Bacteriophage T4↗

PubMed1995

Bait region-thiol ester mapping in human alpha 2-macroglobulin.

The separations between aromatic residues in the bait region and nitroxide spin labels attached to the thiol ester-forming residues (Cys949 and Gln952) in human alpha 2-macroglobulin (alpha 2M) have been determined from paramagnetic broadening effects of the spin labels on bait region 1H NMR signals. We found that both the Cys949 and Gln952 residues are within 11-17 A of the aromatic residues in the bait region, with closer approach of some residues to the Gln952 spin label than to the spin label attached to Cys949. A model of the location of bait regions and thiol esters within an alpha 2M half-molecule is proposed that places the bait regions in the central region of alpha 2M at the interface between the subunits.

Electron Spin Resonance Spectroscopy↗

PubMed1995

Integration of a K+ channel-associated peptide in a lipid bilayer: conformation, lipid-protein interactions, and rotational diffusion.

The 26-residue peptide of sequence KEALYILMVLGFFGFFTLGIMLSYIR, which contains the single putative transmembrane domain of a small protein that is associated with slow voltage-gated K+ channels, has been incorporated in bilayers of dimyristoylphosphatidylcholine by dialysis from 2-chloroethanol to form complexes of homogeneous lipid/peptide ratio. Fourier transform infrared spectroscopy indicates that the peptide is integrated in the lipid bilayer wholly in a beta-sheet conformation. The electron spin resonance spectra of spin-labeled lipids in the lipid/peptide complexes contain a component corresponding to lipids whose chains are motionally restricted in a manner similar to those of lipids at the hydrophobic surface of integral transmembrane proteins. From the dependence of the lipid spin label spectra on the lipid/peptide ratio of the complexes, it is found that ca. 2.5 lipids per peptide monomer, independent of the species of spin-labeled lipid, are motionally restricted by direct interaction with the peptide in the bilayer. This value would be consistent with, e.g., a beta-barrel structure for the peptide in which the beta-strands either are strongly tilted or have a reverse turn at their center. A preferential selectivity of interaction with the peptide is observed for the negatively charged spin-labeled lipids phosphatidic acid, stearic acid, and phosphatidylserine, which indicates close proximity of the positively charged residues at the peptide termini to the lipid headgroups. The saturation-transfer electron spin resonance spectra of the peptide spin-labeled at a cysteine residue replacing Leu18 evidence rather slow rotational diffusion in the lipid complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

A novel electron paramagnetic resonance spin label and its application to study the cross-bridge cycle.

We have used a novel alpha-iodoketone spin-label (IKSL) to study myosin head orientation and cross-bridge dynamics in the putative pre-powerstroke state. Possible perturbation of the cross-bridge cycle by the label was assayed by the sinusoidal analysis method (Kawai and Brandt, 1980; Kawai and Zhao, 1993), which determines the rate constants of the elementary steps in the cycle. A comparison of the rates obtained from unlabeled and IKSL fibers revealed small (10-20%) changes in the ATP hydrolysis rate and in the rate constants of the elementary steps. The labeling induced small changes (< 13%) in the distribution of the cross-bridges among the various intermediate states. Pre-powerstroke cross-bridges were induced by aluminum fluoride in the presence of Ca2+ and ATP. In this state, force development is inhibited, but a large proportion (40%) of the cross-bridges are still attached to the thin filament. We have used conventional electron paramagnetic resonance to measure orientation, and have found that the pre-powerstroke heads are as disordered as in relaxation. Their mobility, as determined by saturation transfer electron paramagnetic resonance, was significantly restricted. Assuming that stiffness is proportional to the fraction of strongly attached heads, the rotational correlation time of the bound heads is estimated to be tau r = approximately 150-400 microseconds.

Aluminum Compounds↗

PubMed1994

Myosin catalytic domain flexibility in MgADP.

Conventional EPR studies of muscle fibers labeled with a novel alpha-iodoketo spin label at Cys-707 of the myosin head revealed substantial internal domain reorganization on the addition of ADP to rigor fibers. The spin probes that are well-ordered in the rigor state become disordered and form two distinct populations. These orientational changes do not correspond to rotation of the myosin catalytic domain as a whole because other probes (maleimide and iodoacetamide nitroxides attached to the same Cys-707 of myosin head) report only a small (5-10 degrees) torsional rotation and little or no change in the tilt angle [Ajtai et al. (1992) Biochemistry 31, 207-17; Fajer (1994) Biophys. J. 66, 2039-50]. In the presence of ADP, the labeled domain becomes more flexible and executes large-amplitude microsecond motions, as measured by saturation-transfer EPR with rates (tau r = 150 microseconds) intermediate between the rotations of detached (tau r = 7 microseconds) and rigor heads (tau r = 2500 microseconds). This finding contrasts with an absence of global motion of the myosin head in ADP (tau r = 2200 microseconds) as reported by the maleimide spin label. Our results imply that the myosin head in a single chemical state (AM.ADP) is capable of attaining many internal configurations, some of which are dynamic. The presence of these slow structural fluctuations might be related to the slow release of the hydrolysis products of actomyosin ATPase.

Adenosine Diphosphate↗

PubMed1994

Time-resolved detection of structural changes during the photocycle of spin-labeled bacteriorhodopsin.

Bacteriorhodopsin was selectively spin labeled at residues 72, 101, or 105 after replacement of the native amino acids by cysteine. Only the electron paramagnetic resonance spectrum of the label at 101 was time-dependent during the photocycle. The spectral change rose with the decay of the M intermediate and fell with recovery of the ground state. The transient signal is interpreted as the result of movement in the C-D or E-F interhelical loop, or in both, coincident with protonation changes at the key aspartate 96 residue. These results link the optically characterized intermediates with localized conformational changes in bacteriorhodopsin during the photocycle.

Bacteriorhodopsins↗

PubMed1994

Site-specific incorporation of biophysical probes into proteins.

Biophysical probes which can detect structural changes in proteins and the interaction of proteins with other macromolecules are important tools in studying protein function. Many difficulties remain, however, in introducing probes into proteins site-specifically. Here we report the successful site-specific incorporation of a spin-labeled, a fluorescent, and a photoactivatible amino acid into a variety of surface and internal sites in bacteriophage T4 lysozyme by using unnatural amino acid mutagenesis. In addition, we report the purification and spectral characterization of T4 lysozyme mutants containing the spin-labeled amino acid and the fluorescent amino acid. The ability to incorporate these probes site-specifically allows for novel studies of protein structure and dynamics. Moreover, this work demonstrates that the Escherichia coli protein biosynthetic machinery can tolerate unnatural amino acids with little resemblance to the natural amino acids.

Bacteriophage T4↗

PubMed1994

Influence of poly(ethylene glycol) and aqueous viscosity on the rotational diffusion of membranous Na,K-ATPase.

The Na,K-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), E.C. 3.6.1.37] in native membranes from the salt gland of Squalus acanthias has been spin-labeled covalently with a chloromercuri nitroxide derivative, and the rotational diffusion of the protein has been studied, as a function of the concentration of glycerol or poly(ethylene glycol) in the suspending medium, by means of saturation-transfer electron spin resonance spectroscopy. The effective rotational correlation time of the protein increases linearly with the viscosity of the aqueous glycerol medium, with a gradient whose value indicates that ca. 50-70% of the volume of the Na,K-ATPase protein is external to the membrane. The effective rotational correlation times of the protein in poly(ethylene glycol) solutions are considerably greater than those in glycerol solutions of the same viscosity and increase nonlinearly with the viscosity of the suspending medium, indicating that increasing concentrations of poly(ethylene glycol) induce aggregation of the integral proteins within the membrane. The value reached at 50% poly(ethylene glycol) corresponds to a degree of aggregation of the proteins between 2 and 5 depending on whether the ethylene glycol polymer is excluded from the membrane surface region. The results are discussed with respect to hydration forces and poly(ethylene glycol)-induced cell fusion.

Animals↗

PubMed1993

Photoactivated conformational changes in rhodopsin: a time-resolved spin label study.

Rhodopsin has been selectively spin-labeled near the cytoplasmic termini of helices C and G. Photoactivation with a light flash induces an electron paramagnetic resonance spectral change in the millisecond time domain, coincident with the appearance of the active metarhodopsin II intermediate. The spectral change is consistent with a small movement near the cytoplasmic termination of the C helix and reverses upon formation of the MIII state. These results provide an important link between the optical changes associated with the retinal chromophore and protein conformational states.

Amino Acid Sequence↗

PubMed1993

Maleimide, iodoacetamide, indanedione, and chloromercuric spin label reagents with derivatized nitroxide rings as ESR reporter groups for protein conformation and dynamics.

The syntheses of eight nitroxide spin labels which bear maleimide, iodoacetamide, indanedione, or chloromercuric reactive groups and, in addition, a second substituent in the nitroxide ring are presented. The second substituent groups range from hydrophobic and hydrophilic esters to carboxylic acid and secondary and tertiary amine groups. The resulting spin labels are characterized with respect both to protein covalent modification and to the electron spin resonance spectral properties of the bound labels. The effect of the various substituents in the spin label on the reactivity toward the membrane-bound shark rectal gland and pig kidney Na,K-ATPase is described. The spectral differences between immobilized and mobile groups observed by electron spin resonance for the different protein-bound spin labels show that, by selecting an appropriate derivative for modification, a large range of different motional sensitivities of the reporter group can be obtained. Such different series of spin labels should therefore be useful for detecting mobility changes arising from conformational transitions in proteins by conventional electron spin resonance spectroscopy or for measurement of protein rotational diffusion using saturation transfer electron spin resonance spectroscopy. The chloromercuric series is found to be particularly useful because of the high reactivity, the lack of reversibility that potentially is associated with the Michael addition reaction, and the wide range of rotational mobility that is exhibited by the different derivatives.

Animals↗

PubMed1993

Saturation transfer electron parametric resonance of an indane-dione spin-label. Calibration with hemoglobin and application to myosin rotational dynamics.

We have used a recently synthesized indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione (InVSL) to study the rotational dynamics of myosin, with saturation-transfer electron paramagnetic resonance (ST-EPR). To determine effective rotational correlation times (tau effr) from InVSL spectra, reference spectra corresponding to known correlation times (tau r) were obtained from InVSL-hemoglobin undergoing isotropic rotational motion in aqueous glycerol solutions. These spectra were used to generate plots of spectral parameters vs. tau r. These plots should be used to analyze ST-EPR spectra of InVSL bound to other proteins, because the spectra are different from those of tempo-maleimide-spin-labeled hemoglobin, which have been used previously as ST-EPR standards. InVSL was covalently attached to the head (subfragment-1; S1) of myosin. EPR spectra and K/EDTA-ATPase activity showed that 70-95% of the heads were labeled, with > or = 90% of the label bound to either cys 707 (SH1) or cys 697 (SH2). ST-EPR spectra of InVSL-S1 attached to glass beads, bound to actin in myofibrils, or precipitated with ammonium sulfate indicated no submillisecond rotational motion. Therefore, InVSL is rigidly immobilized on the protein so that it reports the global rotation of the myosin head. The ST-EPR spectra of InVSL-myosin monomers and filaments indicated tau effr values of 4 and 13 microseconds, respectively, showing that myosin heads undergo microsecond segmental rotations that are more restricted in filaments than in monomers. The observed tau effr values are longer than those previously obtained with other spin labels bound to myosin heads, probably because InVSL binds more rigidly to the protein and/or with a different orientation. Further EPR studies of InVSL-myosin in solution and in muscle fibers should prove complementary to previous work with other labels.

Amino Acid Sequence↗

PubMed1992

Analysis of thiol-topography in Na,K-ATPase using labelling with different maleimide nitroxide derivatives.

Spin-label EPR spectroscopy of shark rectal gland Na,K-ATPase modified at cysteine residues with a variety of maleimide-nitroxide derivatives is used to characterize the different classes of sulphydryl groups. The spin-labelled derivatives vary with respect to charge and lipophilicity, and the chemical reactivity towards modification and inactivation of the Na,K-ATPase is dependent on these properties. Ascorbate is used to reduce the spin-labels in situ, and the kinetics of reduction of the protein-bound spin-labels are found also to depend on the nature of the maleimide-nitroxide derivative. The Na,K-ATPase is labelled either at Class I groups (with retention of enzymatic activity) or at Class II groups (where the enzymatic activity is lost). Although Class I groups are labelled more readily than are Class II groups they are only slightly more susceptible to reduction by ascorbate than the Class II groups, indicating no major difference in environment. The spectral difference observed between immobilized and mobile spin-labels with both Class I and Class II groups labelling is not reflected in widely different reduction kinetics for these two spectral components. Solubilization of the enzyme in an active form does not change the protein structure in terms of increased accessibility of the SH-groups to reduction by ascorbate. The results are discussed in terms of the location of the different SH-groups and the origins of the differences in mobility evident in the EPR spectra of the spin-labelled SH-groups.

Animals↗

PubMed1992

Conventional and saturation transfer EPR spectroscopy of Na+/K(+)-ATPase modified with different maleimide-nitroxide derivatives.

The membranous Na+/K(+)-ATPase from Squalus acanthias has been covalently modified on either Class I or Class II sulphydryl groups using derivatives of 3-(maleimidomethyl)-1-oxyl-2,2,5,5-tetramethylpyrrolidine with substituents of different charge and hydrophobicity attached at the remaining unsubstituted position of the pyrrolidine ring. The substituent groups used were a methyl and a hexyl ester, and di- and tri-methylammonium ethyl esters, as well as the parent underivatized compound. Additionally, another series of maleimide-nitroxides differing (by zero to seven intervening atoms) in the length of the linking group between the maleimide and the pyrrolidine moieties was used. The sites of attachment have been characterized in terms of the rotational mobility and environmental polarity by using conventional and saturation transfer EPR spectroscopy of these spin-labelled reagents. This provides a further sub-classification of the primary Class I and Class II SH-groups on the alpha-subunit of the enzyme, which differ both in their reactivity and influence on the Na+/K(+)-ATPase activity.

Animals↗

PubMed1992

Interfacial ionization and partitioning of membrane-bound local anesthetics.

Consideration of the interfacial protonation equilibria of membrane-associated amphiphiles indicates that the partition coefficients of the protonated and unprotonated species will differ considerably. The partition coefficients of the charged and uncharged forms of spin-labelled myristic acid in dimyristoylphosphatidylcholine bilayer dispersions have been measured by EPR spectroscopy and found to be approximately 140-fold higher for the protonated acid than for the dissociated salt form. This ratio of partition coefficients is found to be in good agreement with that predicted from the interfacial shift in pKa of the fatty acid on its partitioning into the membrane. The latter was determined from the changes in the EPR spectra of the membrane-associated fatty acid with pH and was found to be +2.1 pH units. The interfacial shifts in pKa for a series of spin-labelled analogues of tertiary amine local anaesthetics have been determined from the pH dependence of the partition coefficients in dimyristoylphosphatidylcholine bilayer dispersions and are found mostly to be in the range of approx. -1.0 to -1.5 pH units, corresponding to a 10- to 30-fold higher partition coefficient of the uncharged base compared with that of the charged ammonium form.

Anesthetics↗

PubMed1992

Protein sulfhydryls are protected from irreversible oxidation by conversion to mixed disulfides.

Protein mixed thioselenides formed by reaction of sarcoplasmic reticulum (SR) with diselenide biradical spin labels were quantified by ESR. Whereas the reaction of SR membranes with the diselenide spin label led to a large ESR signal of the unbound monoselenide at equilibrium, treatment of the reaction mixture with a few millimolar hydrogen peroxide converted all of the nitroxides to protein-bound thioselenides. This technique of spin-labeling protein thiols avoids the need to remove unreacted spin labels. The bound spin labels were removable by reduction with excess mercaptoethanol, indicating a specific and reversible labeling of protein thiols. SR that had been extensively labeled with the diselenide spin label was resistant to ATPase inactivation by potent oxidants that arise when myoglobin reacts with hydroperoxides. Unmodified SR lost all activity within 10 min of exposure to either 1 mM tert-butyl hydroperoxide in the presence of 200 microM equine myoglobin or to 100 mM hydrogen peroxide in the absence of myoglobin. In both cases the loss of activity could not be reversed by subsequent treatment with mercaptoethanol. On the other hand, membranes that had been extensively treated with the diselenide spin label and were then subjected to these peroxide treatments were fully active after mercaptoethanol-mediated cleavage of the thioselenides. ESR analysis of spin-labeled SR showed no detectable oxidative cleavage of the thioselenide bonds. Sodium dodecyl sulfate gel electrophoresis showed that peroxide-mediated crosslinking of ATPase observed in unmodified SR membranes did not occur in the diselenide-modified SR membranes. Only limited protection was observed when SR pretreated with glutathione disulfide was incubated with hydroperoxides. In this case, however, the degree of protection was greatly increased when the reaction with glutathione disulfide was carried out in the presence of the supernatant of centrifuged rat liver homogenate, consistent with an acceleration of mixed disulfide formation by a factor tentatively identified as thiol transferase. It is concluded that conversion of protein thiol residues to either thioselenides or mixed disulfides confers protection against irreversible peroxide-dependent oxidation. We suggest that mixed disulfide formation by thiol transferase activity may help protect protein thiols from irreversible oxidation by heme-activated hydroperoxides.

Adenosine Triphosphatases↗

PubMed1991

Contrast agents for magnetic resonance spectroscopy: a method to obtain increased information in in vivo and in vitro spectroscopy.

The concept of contrast, which now is an integral part of many magnetic resonance imaging studies, can be extended successfully to magnetic resonance spectroscopy. It involves the use of paramagnetic molecules whose distribution is restricted in some manner, thereby causing differential effects on the NMR spectra. As an illustrative example, the effects of lipophilic nitroxide stable free radicals on the NMR spectra of serum and lipoproteins are shown. These nitroxides differentially broaden away components of the spectra due to the nuclei of methylene and methyl groups, which enables the usually obscured peaks of lactate to be observed fully. The concept can be applied to differential distribution of the contrast agent on the basis of solubility, charge, and/or compartmentalization. It can be used with any type of NMR spectroscopy and any type of paramagnetic contrast (broadening) agent.

Animals↗