Orientation Dependence of the Electron Spin Resonance Spectrum of Di-<i>t</i>-butyl Nitroxide

Libertini, Louis J.; Griffith, O. Hayes

doi:10.1063/1.1674181

Cited by 139 publications

(52 citation statements)

References 10 publications

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“…The effect of the slightly restricted motion was accounted for by introducing different line widths for each of M, components of the 14N. The second spectrum, corresponding to MG, was taken as an anisotropic powder pattern with both g and A anisotropies (33). In this case, a single value for the line width was used for all M, components.…”

Section: Resultsmentioning

confidence: 99%

Two-state transition between molten globule and unfolded states of acetylcholinesterase as monitored by electron paramagnetic resonance spectroscopy.

Kreimer

Szosenfogel

Goldfarb

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Cys-231 of Torpedo caIlfornica acetyicholinesterase (EC 3.1.1.7) was selectively labeled with the mercury derivative of a stable ntxyl radical. In 1.5 M ganiinlum chloride, this conjugate exists in a molten globule state (MG), whereas in 5 M denaturant, it is in an unfolded state (U). The transtion between the two states is reversible. In the MG, the label is highly immobilize, whereas in the U, it is almost freely rotating. The clearly distinct electron paramagetic resonance (EPR) spectra of the two states permits the study of this transition. Upon elevating the guamdnlum chloride concentration, a decrease in the EPR sinal of the MG occurs concomitantly with an increase in the U signal, the total intensity of the EPR spectra remaining ant. This behavior is characteristic of a two-state tasition. The (4,5,8,9), the description of the MG =. U transition is still controversial. It has been theoretically suggested that, depending on their amino acid compositions, proteins may undergo this transition either by gradual expansion of the polypeptide chain or as a two-state process (10). Others have claimed that it must occur through gradual swelling of MG (11).Similar controversy also exists with respect to the experimental data. Exclusion chromatography suggested that the guanidine hydrochloride (Gdn-HCl)-induced MG = U transition of bovine a-lactalbumin is two state (12). However, an NMR study, published at the same time, presented evidence that the urea-induced MG = U transition of the same protein is noncooperative (13 Size-exclusion chromatography can permit separation of such coexisting species (12, 17); however, this method requires that the rate of interconversion is slow on the time scale of the chromatographic procedure (5, 17). Application of NMR to monitor the MG -Utransition seems promising, since it permits, in principle, simultaneous observation of signals corresponding to MG and U species. However, no separation between MG and U signals was observed for bovine a-lactalbumin in the transition region as a result offast exchange on the NMR time scale (13).Nitroxyl radicals provide spin labels for proteins whose mobility displays high sensitivity to conformational, change (18). Changes in mobility of the bound probe are reflected in concomitant changes in field position and line width (19).Such spectral shifts are usually rather large, >107 sec-1 (in frequency units), whereas rates of interconversion between MG and U are in the range of 1-103sO-1(5, 20), which is slow on an electron paramagnetic resonance (EPR) time scale. Their spectral resolution should thus be feasible. We have shown (21) that a stable nitroxyl radical can be covalently attached to a single cysteine residue, Cys-2231, of Torpedo californica acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7; AcChoEase). We here use this approach for selective modification of AcChoEase with the thiol-specific spin-labeled organomercurial 2,2,5,5-tetramethyl-4-(2-chloromercuriphenyl)-3-imidazoline-1-oxyl (HgR).By using EPR spectrosc...

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Section: Resultsmentioning

confidence: 99%

Two-state transition between molten globule and unfolded states of acetylcholinesterase as monitored by electron paramagnetic resonance spectroscopy.

Kreimer

Szosenfogel

Goldfarb

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…Axial symmetry of g and A tensors (g xx Å g yy , data. The first spectrum was obtained from a dose of 100 L A xx Å A yy ) (14) and spatial random distribution of molecular DTBN. This corresponds formally to 20 layers, if the growth orientations have been assumed for the simulations.…”

Section: Introduction Experimentalmentioning

confidence: 99%

ESR and TPD Investigations of the Adsorption of Di-tert-butyl Nitroxide on Au(111) and NiO(111). Evidence for Long-Range Interactions

Katter

Risse

Schlienz

et al. 1997

Journal of Magnetic Resonance

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“…In electron paramagnetic resonance (EPR) spectroscopy, spectral simulations have been developed by a variety of approaches, including polar coordinates (2,3), spherical harmonics (4), and Eulerian transformations (5). Simulations can be fitted to experimental spectra employing MarquardtLevenberg least-squares routines (6) or the SIMPLEX algorithm (7).…”

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confidence: 99%

Determination of spin-label orientation within the myosin head.

Fajer

1994

Proc. Natl. Acad. Sci. U.S.A.

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Current methods of analyzing EPR spectra of spin-labeled muscle fibers allow the determination of spin-label orientation within the fiber, rather than the orientation of the myosin head itself. In order to describe the orientational distribution of spin labeled myosin heads within the muscle fibers, the orientation of the spin label within the myosin head must be known. The iodoacetamide label orientation in the myosin head was determined to be (16.8, 28.30, 4.2) or (16.6, 72.0, 4.30). These Eulerian angles were obtained from the analysis of EPR spectra of fibers decorated with labeled myosin heads in the absence of ATP, with the assumption that the head's tilt angle is 40°, as observed in a recent EM study [Pollard, T., Bbandari, D., Maupin, P., WachsstockD., Weeds, A. & Zot, H. (1993)Biophys. J. 64, 454-471]. Knowledge ofspin-label orientation will allow for qmntitative determination of myosin head orientation in the various states of the contractile cycle.Since the initial formulation ofthe rotating crossbridge theory (1), much effort has been expended on finding direct evidence for reorientation of the myosin head interacting with actin during muscle contraction. Rotation of the head is postulated to result in strain between the actin and myosin filaments, which is then relieved by the sliding ofthe filaments past each other. Structural studies have involved electron microscopy (EM), x-ray diffraction, and a variety of spectroscopic techniques sensitive to rotational motion. Each of these approaches has different limitations due to the complexity of data interpretation and artifacts due to sample preparation. Spectroscopy, magnetic or optical, has the major advantages of superior sensitivity to orientational changes and of site specificity-i.e., the ability to probe a single site on the protein. There is, however, a problem in translating spectroscopic signals into molecular terms. The problem can be broken into three separate topics: (i) development of simulation algorithms, (ii) optimization techniques to fit simulations to the observed spectra, and (iii) translating the orientational distributions of probes into the orientation of the molecules.In electron paramagnetic resonance (EPR) spectroscopy, spectral simulations have been developed by a variety of approaches, including polar coordinates (2, 3), spherical harmonics (4), and Eulerian transformations (5). Simulations can be fitted to experimental spectra employing MarquardtLevenberg least-squares routines (6) or the SIMPLEX algorithm (7). Alternatively, the orientational distribution of the spins can be determined by direct deconvolution of the spectral lineshape, rather than by fitting (8).Little attempt has been made in the area of relating the spin distribution to the molecular orientation. The stumbling block is the prerequisite of knowing the probe orientation within the molecule. Sometimes it is possible to determine the probe orientation from a single crystal of a protein by correlating the spin axis with the crystallographic axes (9, ...

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Orientation Dependence of the Electron Spin Resonance Spectrum of Di-t-butyl Nitroxide

Cited by 139 publications

References 10 publications

Two-state transition between molten globule and unfolded states of acetylcholinesterase as monitored by electron paramagnetic resonance spectroscopy.

Two-state transition between molten globule and unfolded states of acetylcholinesterase as monitored by electron paramagnetic resonance spectroscopy.

ESR and TPD Investigations of the Adsorption of Di-tert-butyl Nitroxide on Au(111) and NiO(111). Evidence for Long-Range Interactions

Determination of spin-label orientation within the myosin head.

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