Relaxation Time Determinations by Progressive Saturation EPR: Effects of Molecular Motion and Zeeman Modulation for Spin Labels

Лившиц, В. А.; Páli, Tibor; Marsh, Derek

doi:10.1006/jmre.1998.1434

Cited by 24 publications

(33 citation statements)

References 28 publications

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“…(18)). Also for this reason, the effective values obtained for T 1 are lower than the intrinsic values of T 1 determined in comparable systems by saturation recovery EPR (14). It will be noted that the accuracy of determining effective T 1 -values by this method depends essentially only on the accuracy of the determination of the absolute H 1 -values, but requires precise setting of the modulation phase.…”

Section: Experimental Dependence Of Vј 1 -Lineshape On Hmentioning

confidence: 81%

“…In general, they are smaller than the intrinsic values determined by timedomain methods such as saturation recovery (8,9). This point has been dealt with in some detail in a previous publication (14). The application of CW methods with spin labels is, however, restricted mostly to determining spin-lattice relaxation enhancements, rather than absolute values of T 1 .…”

Section: Introductionmentioning

confidence: 97%

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Spin Relaxation Measurements Using First-Harmonic Out-of-Phase Absorption EPR Signals

Лившиц

Páli

Marsh

1998

Journal of Magnetic Resonance

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The dependence on spin-lattice (T1) relaxation of the first-harmonic absorption EPR signal (V'1) detected in phase quadrature with the Zeeman modulation has been investigated both theoretically and experimentally for nitroxide spin labels. Spectral simulations were performed by iterative solution of the Bloch equations that contained explicitly both the modulation and microwave magnetic fields (T. Páli, V. A. Livshits, and D. Marsh, 1996, J. Magn. Reson. B 113, 151-159). It was found that, of the various non-linear EPR displays, the first-harmonic out-of-phase V'1-signal, recorded under conditions of partial saturation of the microwave absorption, is particularly favorable for determining spin-lattice relaxation enhancements because of its superior signal intensity and relative insensitivity to spin-spin (T2) relaxation. By varying the Zeeman modulation frequency it is also possible to tune the optimum sensitivity of the V'1-signal to different ranges of the T1-relaxation time. A Zeeman modulation frequency of 25 kHz appears to be particularly suited to spin label applications. Calibrations are given for the dependence on T1-relaxation time of both the amplitude and the second integral of the V'1-signal recorded under standard conditions. Experiments on different spin labels in solution and in membranes demonstrate the practical usable sensitivity of the V'1-signal, even at modulation frequencies of 25 kHz, and these are used to investigate the dependence on microwave field intensity, in comparison with theoretical predictions. The practicable sensitivity to spin-lattice relaxation enhancements is demonstrated experimentally for a spin-labeled membrane system in the presence of paramagnetic ions. The first-harmonic out-of-phase V'1-signal appears to be the non-linear CW EPR method of choice for determining T1-relaxation enhancements in spin-labeled systems.

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Section: Experimental Dependence Of Vј 1 -Lineshape On Hmentioning

confidence: 81%

Section: Introductionmentioning

confidence: 97%

Spin Relaxation Measurements Using First-Harmonic Out-of-Phase Absorption EPR Signals

Лившиц

Páli

Marsh

1998

Journal of Magnetic Resonance

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“…[10] and [11], the value of V 1,1 is obtained from the solution of Eq. [8], which is expressed as I ᠬ 1 ϭ (U 1,1 , V 1,1 , Z 1,1 ).…”

Section: Theorymentioning

confidence: 99%

Spin Relaxation Measurements Using First-Harmonic Out-of-Phase Absorption EPR Signals: Rotational Motion Effects

Лившиц

Marsh

2000

Journal of Magnetic Resonance

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A recent survey of nonlinear continuous-wave (CW) EPR methods revealed that the first-harmonic absorption EPR signal, detected 90 degrees out of phase with respect to the Zeeman modulation (V(1)(')-EPR), is the most appropriate for determining spin-lattice relaxation enhancements of spin labels (V. A. Livshits, T. Páli, and D. Marsh, 1998, J. Magn. Reson. 134, 113-123). The sensitivity of such V(1)(')-EPR spectra to molecular rotational motion is investigated here by spectral simulations for nitroxyl spin labels, over the entire range of rotational correlation times. Determination of the effective spin-lattice relaxation times is less dependent on rotational mobility than for other nonlinear CW EPR methods, especially at a Zeeman modulation frequency of 25 kHz which is particularly appropriate for spin labels. This relative insensitivity to molecular motion further enhances the usefulness of the V(1)(')-EPR method. Calibrations of the out-of-phase to in-phase spectral intensity (and amplitude) ratios are given as a function of spin-lattice relaxation time, for the full range of spin-label rotational correlation times. Experimental measurements on spin labels in the slow, intermediate, and fast motional regimes of molecular rotation are used to test and validate the method.

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“…Slight deviations are found in the relative values of (T 1 T 2 ) eff compared with those of I ST , because the former depend not only on T 1 eff but also on T 2 eff (i.e., additionally on the line widths). In particular, the larger values of (T 1 T 2 ) eff for the DMPG/cyt ox complexes imply slower rates of chain rotational motion, as is predicted theoretically for correlation times greater than τ R ≈ 10 -8 s (39). The values of (T 1 T 2 ) eff for DMPG/cyt ox complexes in the presence and absence of azide are given in Table 3 for the 5-and 14-PGSL spin labels.…”

Section: Resultsmentioning

confidence: 53%

Nonlinear Electron Paramagnetic Resonance Studies of the Interaction of Cytochrome c Oxidase with Spin-Labeled Lipids in Gel-Phase Membranes

et al. 2000

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The interaction of lipids, spin-labeled at different positions in the sn-2 chain, with cytochrome c oxidase reconstituted in gel-phase membranes of dimyristoylphosphatidylglycerol has been studied by electron paramagnetic resonance (EPR) spectroscopy. Nonlinear EPR methods, both saturation transfer EPR and progressive saturation EPR, were used. Interaction with the protein largely removes the flexibility gradient of the lipid chains in gel-phase membranes. The rotational mobility of the chain segments is reduced, relative to that for gel-phase lipids, by the intramembranous interaction with cytochrome c oxidase. This holds for all positions of chain labeling, but the relative effect is greater for chain segments closer to the terminal methyl ends. Modification of the paramagnetic metal-ion centers in the protein by binding azide has a pronounced effect on the spin-lattice relaxation of the lipid spin labels. This demonstrates that the centers modified are sufficiently close to the first-shell lipids to give appreciable dipolar interactions and that their vertical location in the membrane is closer to the 5-position than to the 14-position of the lipid chains.Cytochrome c oxidase is the terminal member of the mitochondrial respiratory chain that effects the reduction of molecular oxygen to water by means of electrons donated from reduced cytochrome c. The reaction involves pumping protons toward the cytosol to provide the chemiosmotic driving force for ATP synthesis. The cytochrome c oxidase enzyme from beef heart is a multisubunit polytopic transmembrane protein, which contains several metal-ion centers that are designated heme a, heme a 3 , Cu A , and Cu B . The crystal structure of the whole 13-subunit assembly of the bovine enzyme has been determined (1) and further refined recently for different redox forms (2).As a representative large integral membrane protein, cytochrome c oxidase has been the subject of several studies of lipid-protein interactions (3-6) by spin-label EPR 1 methods. The latter has proved to be particularly useful for quantitating the lipid-protein interactions with a wide variety of intrinsic proteins in fluid-phase membranes (for a recent review see ref 7). Far less information is available on lipidprotein interactions in gel-phase lipid membranes. These not only are found in reconstituted systems but also may be considered as generally representative of membrane regions with high lipid packing densities, e.g., in spatially segregated membrane domains (8). The few magnetic resonance studies that concentrate on lipid-protein interactions in gel-phase membranes include 2 H NMR studies of rhodopsin (9) and saturation-transfer electron paramagnetic resonance (ST-EPR) of the myelin proteolipid protein (10) in reconstituted membrane systems. Nonlinear EPR methods (ST-EPR and also progressive saturation EPR) are well suited to such investigations with spin-labeled lipids. This is because both the gel-phase bilayer membrane lipids and the lipids interacting directly with the intramemb...

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Relaxation Time Determinations by Progressive Saturation EPR: Effects of Molecular Motion and Zeeman Modulation for Spin Labels

Cited by 24 publications

References 28 publications

Spin Relaxation Measurements Using First-Harmonic Out-of-Phase Absorption EPR Signals

Spin Relaxation Measurements Using First-Harmonic Out-of-Phase Absorption EPR Signals

Spin Relaxation Measurements Using First-Harmonic Out-of-Phase Absorption EPR Signals: Rotational Motion Effects

Nonlinear Electron Paramagnetic Resonance Studies of the Interaction of Cytochrome c Oxidase with Spin-Labeled Lipids in Gel-Phase Membranes

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