The effects of actin on the electron spin resonance of spin-labeled myosin

Myosin and heavy meromyosin have been spin labeled at either the S₁ or S₂ thiol groups, and their interaction with F-actin has been studied by electron spin resonance, both in the absence of substrate and during the hydrolysis of ATP. The spectrum of myosin labeled at either group indicates strong immobilization of the label. In the absence of substrate, actin added to S₁-labeled myosin slightly increases the separation of the outer spectral peaks, indicating a decrease in the mobility of the spin label. Actin also reduces the microwave power required to saturate the esr signal of S₁-labeled myosin or heavy meromyosin. The latter phenomenon is a more sensitive measure of the actin-myosin interaction than the spectral change seen in the absence of saturation. This suggests that saturation measurements may provide a more sensitive method of detecting changes in the environment of slowly tumbling nitroxide radicals than spectral measurements carried out in the absence of saturation. The decrease in the amplitude of the spectrum on adding actin at saturating microwave power was used to determine the stoichiometry of the interaction between actin and heavy meromyosin. This decrease is maximal when 2 moles of actin monomer are added per mole of heavy meromyosin and is reversed when actin and myosin are dissociated by ATP. During the steady state hydrolysis of ATP, actin had no detectable effect on the spectrum of S₁-labeled myosin. It can be concluded that spin labels bound to the S₁ groups are in a region of the myosin molecule that is affected by the interaction with actin. Actin does not affect the rate at which the bound spin label is reduced by dithiothreitol nor does the spin labeling of S₁ groups affect the activation by actin of the ATPase activity of myosin. These findings suggest that the most likely mechanism by which actin alters the mobility of labels on S₁ groups involves a change in the conformation of myosin. If a spin label is bound to the S₂ thiol groups rather than the S₁ groups, then actin has no detectable effect on the spectrum either in the presence or absence of ATP.     

Heparin modulates conformational states of plasma fibronectin: an electron spin resonance spin label approach

We have examined the interaction between heparin and human plasma fibronectin using electron spin resonance (ESR) spin label methods. The titratable sulfhydryl groups of plasma fibronectin were modified with a maleimide spin label [Lai and Tooney (1984) Arch. Biochem. Biophys. 228, 465-473]. Addition of heparin resulted in a decrease in the maximum splitting value of the ESR spectrum of spin-labeled fibronectin from 66.8 to 64.3 G, suggesting that heparin induces a conformational alteration of plasma fibronectin. This heparin effect was noticeable at a heparin-to-fibronectin ratio of 20 to 1 and reached a plateau at about 100 to 1. Other sulfated carbohydrates were tested; dextran sulfate was found to be as effective as heparin but chondroitin sulfates were ineffective. The results presented suggest that the binding of heparin changes the molecular conformation of plasma fibronectin to a more relaxed or flexible state.     

Radiolysis of spin-labeled DNA: an electron spin resonance investigation

The reactions of free and DNA-bound 2,2,5,5-tetramethylpyrrolidine-N-oxyl (PROXYL) probes with radicals generated during radiolysis of dilute aqueous solutions of DNA were examined. For the free PROXYL probe in deaerated solution with each of the four nucleotides (dAMP, dCMP, dGMP, and TMP) it was found that the pyrimidine radicals were more reactive toward the probe than were the purine radicals. Reactions of the electron adduct of TMP and the hydroxyl radical adducts of dAMP, dGMP, and TMP with the probe resulted in little or no reduction of the probe. For TMP these results are consistent with the fact that both the protonated electron and hydroxyl radical adducts of TMP will covalently bind to the nitroxide function of the probe. Reduction of the PROXYL probe was observed in reactions with the hydroxyl radical adduct of dCMP and with the electron adducts of dAMP, dCMP, and dGMP. Results of the radiolysis of the free PROXYL probe in deaerated dilute solution of DNA suggest that the PROXYL probe protects the DNA from water radical attack as the ratio of DNA bases to PROXYL probe increases above 50:1. Reactions of DNA-bound probes are dependent on the depth of the nitroxide function in relation to the major groove of the DNA helix. Two probes with tether lengths which are less than the depth of the major groove show an expected increase in reactions with DNA base radicals as compared to a probe with a tether that extends beyond the groove. The longer probe is involved largely in reactions with sugar and water radicals along the periphery of the DNA helix. In the presence of oxygen, there is a dramatic decrease in the loss of both the free and DNA-bound probes due to the lack of reaction of these probes with peroxyl radicals formed by the addition of molecular oxygen to DNA radicals.     

Dynamic equilibrium between the two conformational states of spin-labeled tropomyosin

Tropomyosin was labeled with a maleimide nitroxide spin-label attached to cysteine-190 via a succinimido ring which was subsequently opened by incubation at alkaline pH. Electron spin resonance (ESR) spectra showed a temperature-dependent equilibrium, below the main unfolding transition of tropomyosin, between labels which were restricted in their motion (strongly immobilized), predominating at low temperatures, and those which were highly mobile (weakly immobilized), predominating at higher temperatures. These label states were associated with two protein states from a comparison of the ESR spectral changes with the thermal unfolding profile of tropomyosin. The strongly immobilized labels were associated with the completely folded molded and the weakly immobilized labels with a partially unfolded (in the cysteine-190 region) state which is an intermediate in the thermal unfolding of tropomyosin. A spectral subtraction technique was used to measure the concentration ratio of strongly and weakly immobilized labels from which an equilibrium constant, K, was determined at different temperatures. A linear van't Hoff plot was obtained, indicating that the spin-labeled protein is in thermal equilibrium between these two conformational states with delta H = 17 kcal/mol, delta S = 56 cal/(deg X mol), and K = 1.0 at 34 degrees C. An upper limit of 10(7) s-1 for the conformational fluctuation was estimated from the shapes and separation of the two ESR spectral components. In contrast to the label with the opened succinimido ring, the spin-label with an intact succinimido ring remained strongly immobilized on the protein, indicating that in the partially unfolded state the molecule retains structure in the cysteine-190 region.     

Saturation transfer electron paramagnetic resonance of spin-labeled muscle fibers. Dependence of myosin head rotational motion on sarcomere length

We have investigated the orientation and rotational mobility of spin-labeled myosin heads in muscle fibers as a function of the sarcomere length in the absence of ATP. An iodoacetamide spin label was used to label selectively two-thirds of the sulfhydryl-1 groups in glycerinated rabbit psoas muscle. Conventional electron paramagnetic resonance experiments were used to determine the orientation distribution of the probes relative to the fiber axis, and saturation transfer experiments were used to detect sub-millisecond rotational motion. When fibers are at sarcomere length 2.3 microns (full overlap), spin-labeled heads have a high degree of orientational order. The probes are in a single, narrow orientation distribution (full width 15 degrees), and they exhibit no detectable sub-millisecond rotational motion. When fibers are stretched (sarcomere length increased), either before or after labeling, disorder and microsecond mobility increase greatly, in proportion to the fraction of myosin heads that are no longer in the overlap zone between the thick and thin filaments. Saturation transfer difference spectra show that a fraction of myosin heads equal to the fraction outside the overlap zone have much more rotational mobility than those in fibers at full overlap, and almost as much as in synthetic myosin filaments. The most likely interpretation is that some of the probes, corresponding approximately to the fraction of heads in the overlap zone, remain oriented and immobile, while the rest are highly disordered (angular spread greater than 90 degrees) and mobile (microsecond rotational motion). Thus, it appears that myosin heads are rigidly immobilized by actin, but they rotate through large angles on the microsecond time-scale when detached from actin, even in the absence of ATP.     

Electron spin resonance study of light-induced conformational changes in nitroxide labelled bovine rhodopsin

Bovine rhodopsin was isolated in the unbleached form as a retinal disc membrane suspension and spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl. Both conventional and saturation transfer electron spin resonance methods were used to investigate the sensitivity of the spin-label to conformational changes of rhodopsin induced by both transient and long-term exposure to light. The results indicate that the ESR methods do display sensitivity to such changes. An exponential decay curve with a time constant of 10 s was obtained by following the height of a single peak in the saturation transfer electron spin resonance spectrum in response to a single light flash.     

An electron spin resonance study of free radicals from catechol estrogens

Electron spin resonance spectroscopy has been used to demonstrate production of semiquinone free radicals from the oxidation of the catechol estrogens 2- and 4-hydroxyestradiol and 2,6- and 4,6-dihydroxyestradiol. Radicals were generated by horseradish peroxidase/H2O2 or tyrosinase/O2, or by autoxidation, and were detected as their complexes with spin-stabilizing metal ions (Zn2+ and/or Mg2+). Radical production occurs via one- or two-electron oxidation of catechol estrogens, depending on the type of activating system. Autoxidation of catechol estrogens produces superoxide and H2O2 at physiological pH values. The present results also indicate a difference in the reactivity of quinones derived from 2- and 4-hydroxyestradiol. The toxicological significance of these reactions is discussed.     

Selective placement of electron spin resonance spin labels: new structural methods for peptides and proteins.

Electron spin resonance (ESR) is more powerful than ever as a technique for solving biochemical and biophysical problems. Part of the great utility of ESR arises from the use of modern biochemical methods to place spin labels at important positions along the primary sequence of a peptide or protein.     

Dipsticking the major groove of DNA with enzymatically incorporated spin-labeled deoxyuridines by electron spin resonance spectroscopy

Site-specifically spin-labeled deoxyuridine triphosphates with tethers of different lengths were synthesized and then enzymatically incorporated with terminal transferase to form a spin-labeled poly(dT) copolymer. The spin-labeled copolymers were annealed with poly(dA) to form a duplex, which was analyzed by electron spin resonance spectroscopy in a solution of low ionic strength. The spin labels are attached in position 5 of the deoxyuridine and protrude into the major groove. Based on the correlation between tether length of the spin label and the electron spin resonance lineshape, we show that the depth of the major groove of a DNA in its B-form is about 8 A in solution, which is in good agreement with X-ray fiber studies. We also conclude, based on electron spin resonance lineshape simulation data, that the correlation time of the bases in a DNA duplex is of the order of nanoseconds.     

An electron spin resonance study of the reduction of peroxides by anthracycline semiquinones

Direct and spin-trapping electron spin resonance methods have been used to study the reactivity of semiquinone radicals from the anthracycline antibiotics daunorubicin and adriamycin towards peroxides (hydrogen peroxide, t-butyl hydroperoxide and cumene hydroperoxide). Semiquinone radicals were generated by one-electron reduction of anthracyclines, using xanthine/xanthine oxidase. It is shown that the semiquinones are effective reducing agents for all the peroxides. From spin-trapping experiments it is inferred that the radical product is either OH (from H2O2) or an alkoxyl radical (from the hydroperoxides) which undergoes beta-scission to give the methyl radical. The rate constant for reaction of semiquinone with H2O2 is estimated to be approx. 10(4)-10(5) M-1 X s-1. The reduction does not appear to require catalysis by metal ions.     

Interaction of selectively spin-labeled 70S ribosomes with tRNAPhe. An electron paramagnetic resonance study

70S ribosomes from Escherichia coli, selectively spin labeled on the SH groups of proteins S18, S12, S21, S17, and L27, were used to study the formation of the tertiary complex ribosome-poly(U)-tRNAPhe. Most of these ribosomal proteins are located in the region of binding of tRNA. The electron paramagnetic resonance observable structural change suggests a loosening of the ribosome structure upon binding of the tRNA molecule.     

Competitive ion binding to low density lipoproteins: an electron spin resonance study

The electron spin resonance (ESR) technique was used to evaluate binding constants for Ca(II) and Mg(II) in interaction with low density lipoprotein (LDL). The Ca(II) or Mg(II) ions competed with the paramagnetic Mn(II) ions for the same binding sites of two different classes on the LDL surface. For each ion competing with Mn(II), the solutions of eight non-linear competition equations were fit to the experimental titration curves, with two adjustable parameters, the two binding constants. The derived "intrinsic" values (the values corrected for the electrolyte-induced change of the surface potential) for "strong" binding sites for Ca(II) (170 +/- 85 M-1) and Mg(II) (60 +/- 30 M-1) differ significantly from the respective value for Mn(II) (760 M-1). The values for the "weak" binding sites (18 M-1, 15 M-1 and 10 M-1 for Mn(II), Ca(II) and Mg(II), respectively are in the range of the binding constants for these ions in interaction with model membranes.     

An electron spin resonance spin-label study of lipophilin in oriented phospholipid bilayers

Model membranes consisting of dimyristoyl phosphatidylcholine and a hydrophobic protein from bovine myelin, lipophilin, were studied using the cholesterol-resembling cholestane ESR spin label. Orientation of the membranes made it possible to deconvolute the spectra into two fractions, one of oriented spin labels reflecting phospholipid bilayer of high order, and one of isotropically tumbling spin labels ascribed to the lipid fraction surrounding the protein molecule (boundary lipid). This isotropic tumbling is different from the behavior of phospholipid molecules near the protein, which retain some degree of order, and indicates that the boundary lipid fraction in our model system forms a rather fluid environment for the protein. A nonlinear relation was found between protein concentration and amount of boundary spin labels. Addition of cholesterol decreases the amount of boundary spin labels. Both findings form evidence for a preferential binding of cholesterol by the membrane protein.     

Characterization of structural changes in vimentin bearing an epidermolysis bullosa simplex-like mutation using site-directed spin labeling and electron paramagnetic resonance

Mutations in intermediate filament protein genes are responsible for a number of inherited genetic diseases including skin blistering diseases, corneal opacities, and neurological degenerations. Mutation of the arginine (Arg) residue of the highly conserved LNDR motif has been shown to be causative in inherited disorders in at least four different intermediate filament (IF) proteins found in skin, cornea, and the central nervous system. Thus this residue appears to be broadly important to IF assembly and/or function. While the genetic basis for these diseases has been clearly defined, the inability to determine crystal structure for IFs has precluded a determination of how these mutations affect assembly/structure/function of IFs. To investigate the impact of mutation at this site in IFs, we have mutated the LNDR to LNDS in vimentin, a Type III intermediate filament protein, and have examined the impact of this change on assembly using electron paramagnetic resonance. Compared with wild type vimentin, the mutant shows normal formation of the coiled coil dimer, with a slight reduction in the stability of the dimer in rod domain 1. Probing the dimer-dimer interactions shows the formation of normal dimer centered on residue 191 but a failure of dimerization at residue 348 in rod domain 2. These data point toward a specific stage of assembly at which a common disease-causing mutation in IF proteins interrupts assembly.     

An electron spin resonance study of cholestane spin label in aqueous mixtures of biliary lipids.

The effect of cholesterol on the fluidity of the phospholipid matrix in mixed micelles derived from bile salts and lecithin has been determined by the paramagnetic probe technique. It was found that correlation times for the cholestane spin label were discontinuous functions of cholesterol content and that these discontinuities correlate with the equilibrium solubility limit for cholesterol in this quaternary system. The origin of these discontinuities is attributed to the existence of another aggregate in addition to the discshaped mixed micelle in lipid solutions supersaturated with cholesterol.     

Photogeneration of superoxide by adriamycin and daunomycin. An electron spin resonance and spin trapping study

The hydroxyl and superoxide anion spin adducts of DMPO and 4-MePyBN, respectively, were obtained during photoirradiation of adriamycin and daunomycin solutions with visible light. Ethanol and dimethyl sulfoxide did not scavenge hydroxyl radicals in the photoirradiated drug solutions. Furthermore, the hydroxyl-DMPO spin adduct is not formed in the photolysis of air-free drug solutions, indicating that hydroxyl radicals are not directly produced in the photochemical reactions. Instead, the observed hydroxyl-DMPO is formed from the decay of the superoxide anion-DMPO spin adduct. The mechanism for generating the superoxide anion radical appears to be a direct electron transfer from the photoexcited adriamycin and daunomycin to dissolved oxygen.     

Iron and copper binding by fungal phenolic polymers: an electron spin resonance study

In an electron spin resonance (ESR) survey of fungal phenolic polymers and melanins, it was found that the phenolic polymers fromEpicoccum purpurascens andPenicillium funiculosum displayed hyperfine splitting, indicating the presence of transition metal bonding. The ESR parameters agree well with those of iron and copper organic complexes. Further, the copper-complex ESR parameters were similar to those reported for mixed complexes of copper, bipyridyl and dicarboxylic acids; this suggests that nitrogen-containing groups from the proteinaceous moiety are involved in the process. Acid hydrolysis removed the proteins and metals, but the hydrolyzed polymers were still able to remove 72% of the copper present in a 0.5× 10^–4 M solution. It was concluded that fungal phenolic polymers, which constitute a part of the soil humus, may play an important role in the translocation of metal ions and their availability to biological systems.Deceased