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.     

Sensitive detection and localization of hydroxyl radical production in cucumber roots and Arabidopsis seedlings by spin trapping electron paramagnetic resonance spectroscopy

As reactive oxygen species are important for many fundamental biological processes in plants, specific and sensitive techniques for their detection in vivo are essential. In particular, the analysis of hydroxyl radical (OH*) formation in biological reactions has rarely been attempted. Here, it is shown that spin trapping electron paramagnetic resonance (EPR) spectroscopy allows the detection and quantitative estimation of OH* production in vivo in one single cucumber seedling root. It is possible to localize the OH* production site to the growth zone of the root by varying the position of the intact seedling inside the resonator cavity of the EPR spectrometer. Moreover, the demonstration of impaired OH* formation in the root of the Arabidopsis mutant rhd2 impaired in a superoxide-producing Nicotimamide adenine dinucleotide phosphate (NADPH) oxidase has been accomplished. Spin trapping EPR provides a valuable tool for analyzing the production of OH*in vivo with high resolution in small tissue samples.     

High-resolution electron microscopy of helical specimens: a fresh look at tobacco mosaic virus

The treatment of helical objects as a string of single particles has become an established technique to resolve their three-dimensional (3D) structure using electron cryo-microscopy. It can be applied to a wide range of helical particles such as viruses, microtubules and helical filaments. We have made improvements to this approach using Tobacco Mosaic Virus (TMV) as a test specimen and obtained a map from 210,000 asymmetric units at a resolution better than 5 A. This was made possible by performing a full correction of the contrast transfer function of the microscope. Alignment of helical segments was helped by constraints derived from the helical symmetry of the virus. Furthermore, symmetrization was implemented by multiple inclusions of symmetry-related views in the 3D reconstruction. We used the density map to build an atomic model of TMV. The model was refined using a real-space refinement strategy that accommodates multiple conformers. The atomic model shows significant deviations from the deposited model for the helical form of TMV at the lower-radius region (residues 88 to 109). This region appears more ordered with well-defined secondary structure, compared with the earlier helical structure. The RNA phosphate backbone is sandwiched between two arginine side-chains, stabilizing the interaction between RNA and coat protein. A cluster of two or three carboxylates is buried in a hydrophobic environment isolating it from neighboring subunits. These carboxylates may represent the so-called Caspar carboxylates that form a metastable switch for viral disassembly. Overall, the observed differences suggest that the new model represents a different, more stable state of the virus, compared with the earlier published model.     

Characterization of the copper(II) binding site in the pink copper binding protein CusF by electron paramagnetic resonance spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy has been used to structurally characterize the copper-binding site in CusF protein from Escherichia coli. The EPR spectra indicate a single type II copper center with parameters typical for nitrogen and oxygen ligands (A~200 G, g~2.186, g~2.051). The pulsed EPR data show that one of the ligands to Cu²⁺ is an imidazole ring of a histidine residue. The remote amino nitrogen of this imidazole ring is readily observed by electron spin-echo envelope modulation spectroscopy, while the imino nitrogen that is directly coordinated to the Cu²⁺ ion is observed by pulsed electron–nuclear double resonance (ENDOR). In addition, the ENDOR spectra reveal the presence of one more nitrogen ligand that was assigned to be a deprotonated peptide nitrogen. Apart from the two nitrogen ligands, it has been established that there are two nearby hydroxyl protons, although whether these belong to a single equatorial water ligand or two equatorial hydroxide ligands is not known.

Site-directed spin labeling electron paramagnetic resonance study of the ORF1 protein from a mouse L1 retrotransposon

Long interspersed nuclear element-1 is a highly abundant mammalian retrotransposon that comprises 17% of the human genome. L1 retrotransposition requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. ORF1p has been shown to adopt a homo-trimeric, asymmetric dumbbell-shaped structure. However, its atomic-level structure and mechanism of RNA binding remains poorly understood. Here, we report the results of a site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) study of 27 residues within the RNA binding region of the full-length protein. The EPR data are compatible with the large RNA binding lobe of ORF1p containing a RNA recognition motif (RRM) domain and a carboxyl-terminal domain (CTD) that are predicted from crystallographic and NMR studies of smaller fragments of the protein. Interestingly, the EPR data indicate that residues in strands β3 and β4 of the RRM are structurally unstable, compatible with the previously observed sensitivity of this region to proteolysis. Affinity measurements and RNA-dependent EPR spectral changes map the RNA binding site on ORF1p to residues located in strands β3 and β4 of the RRM domain and to helix α1 of the CTD. Complementary in vivo studies also identify residues within the RRM domain that are required for retrotransposition. We propose that in the context of the full-length trimeric protein these distinct surfaces are positioned adjacent to one another providing a continuous surface that may interact with nucleic acids.     

Hydroxyl radical formation in chondrocytes and cartilage as detected by electron paramagnetic resonance spectroscopy using spin trapping reagents

Chondrocytes have been shown to produce superoxide and hydrogen peroxide, suggesting possible formation of hydroxyl radical in these cells. In this study, we used electron spin resonance/spin trapping technique to detect hydroxyl radicals in chondrocytes. We found that hydroxyl radicals could be detected as α-hydroxyethyl spin trapped adduct of 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN) in chondrocytes stimulated with phorbol 12-myristate 13-acetate in the presence of ferrous ion. The formation of hydroxyl radical appears to be mediated by the transition metal-catalyzed Haber-Weiss reaction since no hydroxyl radical was detected in the absence of exogenous iron. The hydroxyl radical formation was inhibited by catalase but not by superoxide dismutase, suggesting that the hydrogen peroxide is the precursor. Cytokines, IL-1 and TNF enhanced the hydroxyl radical formation in phorbol 12-myristate 13-acetate treated chondrocytes. Interestingly, hydroxyl radical could be detected in unstimulated fresh human and rabbit cartilage tissue pieces in the presence of iron. These results suggest that the formation of hydroxyl radical in cartilage could play a role in cartilage matrix degradation.     

Protection of transgenic tobacco plants expressing bovine pancreatic ribonuclease against tobacco mosaic virus

Transgenic tobacco plants (Nicotiana tabacum cv. SR1) expressing extracellular pancreatic ribonuclease from Bos taurus and characterized by an increased level of ribonuclease activity in leaf extracts were challenged with tobacco mosaic virus. The transgenic plants exhibited a significantly higher level of protection against the virus infection than the control non-transformed plants. The protection was evidenced by the absence (or significant delay) of the appearance of typical mosaic symptoms and the retarded accumulation of infectious virus and viral antigen. These results demonstrate that modulation of extracellular nuclease expression can be efficiently used in promoting protection against viral diseases.     

Identification of apical membranes from tight epithelia using spin-labeled amiloride and electron paramagnetic resonance spectroscopy

Summary Apical cell membranes from Na⁺-transporting epithelia were identified in centrifugal fractions prepared from homogenates of rainbow trout kidney, gill and frog skin using a spinlabeled, nitroxide derivative of amiloride and electron paramagnetic resonance spectroscopy. Spin-labeled amiloride (ASp) is a potent inhibitor of Na⁺ transport. Frog skin shortcircuit current was inhibited by 50% in the presence of 7×10^–8 m ASp, whereas 4×10^–7 m amiloride was required to obtain the same effect. ASp is a suitable probe for the amiloride binding site based on analytical criteria: Unbound ASp produces an EPR signal linear with concentration and detectable at micromolar concentrations. Estimates of ASp binding can usually be made on less than 100 g of membrane protein. While ASp binds nonspecifically to many materials, amiloride- or benzamil-displaceable binding occurred only in trout gill and kidney, and in frog skin, but not in trout skeletal muscle. ASp binds to membrane fractions produced by differential centrifugation of trout gill, kidney and frog skin. In trout gill and kidney, 81% and 91%, respectively, of the amiloride-displaceable ASp binding is found in the 10,000 xg fraction. All of the ASp binding in frog skin is found in the 10,000 xg fraction. These data indicate that spin-labeled amiloride is a useful probe for the identification of the amiloride binding site, and electron paramagnetic resonance spectroscopy will allow the amiloride binding site to be used as a molecular marker for apical membranes.     

Quantification of binding of some thiol-reactive clenbuterol analogues to bovine serum albumin by electron paramagnetic resonance spectroscopy

The only free thiol group of bovine serum albumin (BSA) was coupled in a high yield with some novel thiol-reactive clenbuterol analogues. The unreacted cysteines were probed with maleimide spin label to determine the yield of the coupling reaction. A novel approach to determining free thiol groups of BSA quantitatively by electron paramagnetic resonance spectroscopy and spectral decomposition without the usual gel-filtration step or extensive dialysis is presented.     

Mechanism of photoinduced electron transfer in photosystem II reaction centers

The shape of the EPR spectrum of the triplet state of photosystem II reaction centers with a singly reduced primary acceptor complex Q_AFe²⁺ was studied. It was shown that the spectroscopic properties do not significantly change when the relaxation of the primary acceptor is accelerated and when the magnetic interaction between the reduced quinone molecule Q_A and the nonheme iron ion Fe²⁺ is disrupted. This observation confirmed the earlier conclusion that the anisotropy of the quantum yield of the triplet state is the main cause of the anomalous shape of the EPR spectrum. A scheme of primary processes in photosystem II that is consistent with the observed properties of the EPR spectrum of the triplet state is discussed.     

应用RNAi技术培育抗TMV病毒转基因烟草

利用烟草花叶病毒(TMV)外壳蛋白基因构建RNAi干涉载体, 通过叶盘法转化至烟草K326 和龙江911两个栽培品种。对转基因株系的荧光定量PCR分析表明, 不同转基因株系的病毒RNA靶序列都得到一定程度的降解, 抗病性鉴定结果证实, 转基因K326和龙江911两个栽培品种的转基因材料分别有83％和90％转基因株系对TMV呈现免疫级抗性。     

Antioxidant properties of indapamide, 5-OH indapamide and hydrochlorothiazide evaluated by oxygen-radical absorbing capacity and electron paramagnetic resonance

The aim of these experiments was to investigate the radical scavenging properties of three diuretics: indapamide (IND) and its major metabolite, 5-OH indapamide (5-OH IND), compared to a reference diuretic, hydrochlorothiazide (HTZ). Electron Paramagnetic Resonance (EPR) was used to determine the scavenging abilities of these compounds on enzymatically produced superoxide radical anion, with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) used as a spin-trap. These experiments revealed that IND and specially 5-OH IND were effective superoxide radical anion scavengers at 0.2 mg/ml. In the second part of these studies, allophycocyanin was used as an indicator of free radical mediated protein damage. In the assay, 2,2-azobis(2-amidinopropane) hydrochloride (AAPH) was used as a peroxyl radical generator, Trolox (a water-soluble analogue of vitamin E) as a control standard, and the loss of allophycocyanin fluorescence was monitored. The antioxidant effects of the diuretics were expressed in oxygen-radical absorbing capacity (ORAC), where one ORAC unit equals the net protection produced by 1 µM Trolox. HTZ showed no protection up to 100 µM final concentration, whereas IND and 5-OH IND showed linear correlation with respect to concentration when expressed in ORAC units: 5-OH IND induced the highest protection against peroxyl radical. The above observations suggested that IND and 5-OH IND are potent radical scavengers, with the metabolite 5-OH IND having a superior antioxidant potency than IND. By contrast, HTZ had no effect. These radical scavenging properties of 5-OH IND may be of clinical interest for vascular protection and may help to protect the heart from oxidative injury.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Quantized incorporation of RNA during assembly of tobacco mosaic virus from protein disks

Reassembly of tobacco mosaic virus from the isolated RNA and protein, supplied as a disk preparation consisting of over 75% as the disk aggregate, has been followed by the protection of the RNA from nuclease digestion. The sizes of the RNA fragments were determined on agarose/acrylamide gels.During the first few minutes the protected RNA is found to be “quantized” into discrete lengths, differing on average by about 50 or 100 nucleotides, corresponding to one or two turns of the virus helix and strongly supporting the hypothesis that elongation in the major direction, towards the 5′-hydroxyl end, is occurring by the direct addition of protein disks. Protected RNA of the full length found in tobacco mosaic virus is visible within six minutes of starting reassembly, and by 30 minutes most of the RNA is fully protected.     

Saturation transfer electron paramagnetic resonance detection of sickle hemoglobin aggregation during deoxygenation.

In Myxicola axons, substitution of tetramethylammonium (TMA+) for Cs+ alters intramembrane charge movements (gating currents). Although the total charge moved during and following a depolarizing step remains constant, with TMA+ the ON response has additional slower component(s), and the OFF response is retarded. Concommitantly, TMA+ produces the same voltage-dependent block of Na+ inactivation in Myxicola as has been observed in other preparations. At large positive potentials as many as 70% of the Na+ channels fail to inactivate in the steady state. In addition, TMA+ slows Na+ activation, retards the inactivation of those Na+ channels that remain able to inactivate, and decreases the maximum Na+ conductance. The steady-state Na+ conductance induced by internal TMA+ or Na+ is consistent with a scheme in which these internal cations simply modify Na+ channels in an all-or-none fashion so that a fraction become incapable of inactivating.     

Heterogeneity in the structural dynamics of Sulfolobus solfataricus beta-glycosidase revealed by electron paramagnetic resonance and frequency domain fluorometry

The local and global dynamics of the Sulfolobus solfataricus beta-glycosidase were studied by electron spin resonance and time-resolved fluorescence techniques. For electron paramagnetic resonance (EPR) investigations, the protein was covalently modified by the maleimido nitroxide spin label, which is specific for cysteine -SH groups, at position 344 and at position 101, where Ser-101 was changed into a cysteine by site-directed mutagenesis. The greater reactivity of exposed Cys-101 suggested the exclusive modification of this amino acid compared with Cys-344. The labeled proteins underwent temperature perturbation in the range 290-335 K and the values of the spin-label rotation correlation frequencies (nu(c)) ranged from 6 x 10(7) to 2 x 10(8) sec(-1) for the protein labeled at position C344 and from 5.62 x 10(7) to 1.10 x 10(8) sec(-1) for the protein labeled at C101. These rotation correlation values are related to the local dynamic characteristics of the protein matrix. The temperature dependence of rotation correlation frequencies expressed in terms of Arrhenius coordinates (log (nu(c)) vs. 1/T) for the protein labeled at C344 exhibited a linear dependence but with a change in the slope at 311 K. For the protein labeled at C101, no change in the slope was observed at the same temperature. General dynamic information was deduced from the analysis of the fluorescence emission decay of the tryptophanyl residues that are present in each region of the protein structure. Fluorescence data analysis highlighted a bimodal distribution of fluorescence lifetimes arising from the contribution of two emitting groups: one consisting of closely clustered tryptophans responsible for the long-lived emission component (7.1 nsec) and the other composed of tryptophans nearer to the protein surface, which can be associated to the short-lived component (2.5 nsec). The temperature dependence of lifetime distribution parameters linked to the long-lived and short-lived components, expressed in Arrhenius coordinates, showed two different points in which the change in the slope occurred (i.e., 328 K and 338 K, respectively). The Arrhenius analysis of data provided the activation energy relative to the conformational changes characterizing the local and global movements running through the protein matrix.     

Transition metal and organic radical components of carp liver tissue observed by electron paramagnetic resonance spectroscopy

Paramagnetic transition metal centers and organic radicals in liver from wild-type carp (Cyprinus carpio) were characterized by electron paramagnetic resonance (EPR) spectroscopy. Approximately twelve EPR signals were observed at 77 K with resonance positions between g=1.8 and g=2.5. Identification was facilitated by a study of the variation in signal intensity with microwave power (microwave power saturation) for each signal. Many were organic radical or iron signals from typical liver enzymes, including cytochrome P450, coenzyme Q₁₀, NADH dehydrogenase, and succinate dehydrogenase, cytochrome c oxidase and/or catalase. Of special interest were two signals that are not normally found in mammalian liver. The first was a six-line signal from divalent manganese, which was evident in the spectra in quantities suggestive of a functional role. The second was probably a signal from nitrosylated non-heme iron and may be related to the presence of nitrogen-containing compounds produced by nitrifying bacteria in the aquatic environment. These notable differences between the EPR spectra of fish and mammalian liver suggest major metabolic differences between the two systems.     

In vivo detection of tobacco mosaic virus-induced local and systemic oxidative burst by electron paramagnetic resonance spectroscopy

This is the first demonstration that tobacco mosaic virus-induced oxidative stress in a necrotic host plant is signalled by an elevated level of monodehydroascorbate (MDA) radicals detected by electron paramagnetic resonance spectroscopy. Furthermore, systemic acquired resistance induced in remote leaves of Xanthi-nc tobacco is also associated with stimulated MDA signals indicative of a microoxidative burst.     

Denaturation studies of active-site labeled papain using electron paramagnetic resonance and fluorescence spectroscopy.

A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and fluorescence results were in agreement and both paralleled enzymatic activity of papain with respect to both the variation in pH and denaturation. These results strongly suggest that SL-PMB and Acrylodan labels can be used to characterize the physical state of the active site of the enzyme.