Category Archives: Nitroxides

Selective High-Resolution Detection of Membrane Protein-Ligand Interaction in Native Membranes Using Trityl-Nitroxide PELDOR

Joseph, B., et al., Selective High-Resolution Detection of Membrane Protein-Ligand Interaction in Native Membranes Using Trityl-Nitroxide PELDOR. Angew Chem Int Ed Engl, 2016. 55(38): p. 11538-42.

https://www.ncbi.nlm.nih.gov/pubmed/27511025

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero-oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro-electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter-subunit interaction and the intra-subunit conformational changes in hetero-oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein-ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.

Room-temperature electron spin relaxation of nitroxides immobilized in trehalose: Effect of substituents adjacent to NO-group

Kuzhelev, A.A., et al., Room-temperature electron spin relaxation of nitroxides immobilized in trehalose: Effect of substituents adjacent to NO-group. J. Magn. Reson., 2016. 266: p. 1-7.

http://www.sciencedirect.com/science/article/pii/S1090780716001178

Trehalose has been recently promoted as efficient immobilizer of biomolecules for room-temperature EPR studies, including distance measurements between attached nitroxide spin labels. Generally, the structure of nitroxide influences the electron spin relaxation times, being crucial parameters for room-temperature pulse EPR measurements. Therefore, in this work we investigated a series of nitroxides with different substituents adjacent to NO-moiety including spirocyclohexane, spirocyclopentane, tetraethyl and tetramethyl groups. Electron spin relaxation times (T1, Tm) of these radicals immobilized in trehalose were measured at room temperature at X- and Q-bands (9/34 GHz). In addition, a comparison was made with the corresponding relaxation times in nitroxide-labeled DNA immobilized in trehalose. In all cases phase memory times Tm were close to 700 ns and did not essentially depend on structure of substituents. Comparison of temperature dependences of Tm at T = 80–300 K shows that the benefit of spirocyclohexane substituents well-known at medium temperatures (∼100–180 K) becomes negligible at 300 K. Therefore, unless there are specific interactions between spin labels and biomolecules, the room-temperature value of Tm in trehalose is weakly dependent on the structure of substituents adjacent to NO-moiety of nitroxide. The issues of specific interactions and stability of nitroxide labels in biological media might be more important for room temperature pulsed dipolar EPR than differences in intrinsic spin relaxation of radicals.

Dynamics of 4-oxo-TEMPO-d16-(15)N nitroxide-propylene glycol system studied by ESR and ESE in liquid and glassy state in temperature range 10-295K

Goslar, J., S.K. Hoffmann, and S. Lijewski, Dynamics of 4-oxo-TEMPO-d16-(15)N nitroxide-propylene glycol system studied by ESR and ESE in liquid and glassy state in temperature range 10-295K. J Magn Reson, 2016. 269: p. 162-75.

http://www.ncbi.nlm.nih.gov/pubmed/27323281

ESR spectra and electron spin relaxation of nitroxide radical in 4-oxo-TEMPO-d16-(15)N in propylene glycol were studied at X-band in the temperature range 10-295K. The spin-lattice relaxation in the liquid viscous state determined from the resonance line shape is governed by three mechanisms occurring during isotropic molecular reorientations. In the glassy state below 200K the spin-lattice relaxation, phase relaxation and electron spin echo envelope modulations (ESEEM) were studied by pulse spin echo technique using 2-pulse and 3-pulse induced signals. Electron spin-lattice relaxation is governed by a single non-phonon relaxation process produced by localized oscillators of energy 76cm(-1). Electron spin dephasing is dominated by a molecular motion producing a resonance-type peak in the temperature dependence of the dephasing rate around 120K. The origin of the peak is discussed and a simple method for the peak shape analysis is proposed, which gives the activation energy of a thermally activated motion Ea=7.8kJ/mol and correlation time tau0=10(-8)s. The spin echo amplitude is strongly modulated and FT spectrum contains a doublet of lines centered around the (2)D nuclei Zeeman frequency. The splitting into the doublet is discussed as due to a weak hyperfine coupling of nitroxide unpaired electron with deuterium of reorienting CD3 groups.

Matrix-free DNP-enhanced NMR spectroscopy of liposomes using a lipid-anchored biradical

Fernandez-de-Alba, C., et al., Matrix-free DNP-enhanced NMR spectroscopy of liposomes using a lipid-anchored biradical. Chemistry, 2015. 21(12): p. 4512-7.

http://www.ncbi.nlm.nih.gov/pubmed/25663569

Magic-angle spinning dynamic nuclear polarization (MAS-DNP) has been proven to be a powerful technique to enhance the sensitivity of solid-state NMR (SSNMR) in a wide range of systems. Here, we show that DNP can be used to polarize lipids using a lipid-anchored polarizing agent. More specifically, we introduce a C16-functionalized biradical, which allows localization of the polarizing agents in the lipid bilayer and DNP experiments to be performed in the absence of excess cryo-protectant molecules (glycerol, dimethyl sulfoxide, etc.). This constitutes another original example of the matrix-free DNP approach that we recently introduced.

Electron Spin–Lattice Relaxation Mechanisms of Nitroxyl Radicals in Ionic Liquids and Conventional Organic Liquids: Temperature Dependence of a Thermally Activated Process

A detailed understanding of the electron-spin relaxation mechanisms in polarizing agents used for DMP-NMR spectroscopy is crucial for the understanding of the DNP process and to optimize polarizing agents for different DNP applications. The entire study was performed at X-Band frequencies (9 GHz, 14 MHz 1H) and provides many details about the relaxation behavior of nitroxide radicals – important either for low-field ODNP experiments but also very relavant for high-field solution-state DNP experiments.

Kundu, K., et al., Electron Spin–Lattice Relaxation Mechanisms of Nitroxyl Radicals in Ionic Liquids and Conventional Organic Liquids: Temperature Dependence of a Thermally Activated Process. The Journal of Physical Chemistry B, 2015. 119(12): p. 4501-4511.

http://dx.doi.org/10.1021/acs.jpcb.5b00431

During the past two decades, several studies have established a significant role played by a thermally activated process in the electron spin relaxation of nitroxyl free radicals in liquid solutions. Its role has been used to explain the spin relaxation behavior of these radicals in a wide range of viscosities and microwave frequencies. However, no temperature dependence of this process has been reported. In this work, our main aim was to investigate the temperature dependence of this process in neat solvents. Electron spin?lattice relaxation times of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-TEMPO (TEMPOL), in X-band microwave frequency, were measured by the pulse saturation recovery technique in three room-temperature ionic liquids ([bmim][BF4], [emim][BF4], and [bmim][PF6]), di-isononyl phthalate, and sec-butyl benzene. The ionic liquids provided a wide range of viscosity in a modest range of temperature. An auxiliary aim was to examine whether the dynamics of a probe molecule dissolved in ionic liquids was different from that in conventional molecular liquids, as claimed in several reports on fluorescence dynamics in ionic liquids. This was the reason for the inclusion of di-isononyl phthalate, whose viscosities are similar to that of the ionic liquids in similar temperatures, and sec-butyl benzene. Rotational correlation times of the nitroxyl radicals were determined from the hyperfine dependence of the electron paramagnetic resonance (EPR) line widths. Observation of highly well-resolved proton hyperfine lines, riding over the nitrogen hyperfine lines, in the low viscosity regime in all the solvents, gave more accurate values of the rotational correlation times than the values generally measured in the absence of these hyperfine lines and reported in the literature. The measured rotational correlation times obeyed a modified Stokes?Einstein?Debye relation of temperature dependence in all solvents. By separating the contributions of g-anisotropy, A-anisotropy and spin-rotation interactions from the observed electron spin?lattice relaxation rates, the contribution of the thermally activated process was obtained and compared with its expression for the temperature dependence. Consistent values of various fitted parameters, used in the expression of the thermal process, have been found, and the applicability of the expression of the thermally activated process to describe the temperature dependence in liquid solutions has been vindicated. Moderate solvent dependence of the thermally activated process has also been observed. The rotational correlation times and the spin?lattice relaxation processes of nitroxyls in ionic liquids and in conventional organic liquids are shown to be explicable on a similar footing, requiring no special treatment for ionic liquids.

High-field ELDOR-detected NMR study of a nitroxide radical in disordered solids: Towards characterization of heterogeneity of microenvironments in spin-labeled systems

With nitroxides one of the most popular polarizing agents for DNP-NMR spectroscopy, this article gives more insights into the precise interactions (hyperfine, quadrupolar, g anisotropy …) at high magnetic fields.

Nalepa, A., et al., High-field ELDOR-detected NMR study of a nitroxide radical in disordered solids: Towards characterization of heterogeneity of microenvironments in spin-labeled systems. J. Magn. Reson., 2014. 242(0): p. 203-213.

http://dx.doi.org/10.1016/j.jmr.2014.02.026

The combination of high-field EPR with site-directed spin-labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in probing the polarity and proticity characteristics of protein/matrix systems. This information is concluded from the principal components of the nitroxide Zeeman (g), nitrogen hyperfine (A) and quadrupole (P) tensors of the spin labels attached to specific sites. Recent multi-frequency high-field EPR studies underlined the complexity of the problem to treat the nitroxide microenvironment in proteins adequately due to inherent heterogeneities which result in several principal x-components of the nitroxide g-tensor. Concomitant, but distinctly different nitrogen hyperfine components could, however, not be determined from high-field cw EPR experiments owing to the large intrinsic EPR linewidth in fully protonated guest/host systems. It is shown in this work that, using the W-band (95 GHz) ELDOR- (electron–electron double resonance) detected NMR (EDNMR) method, different principal nitrogen hyperfine, Azz, and quadrupole, Pzz, tensor values of a nitroxide radical in glassy 2-propanol matrix can be measured with high accuracy. They belong to nitroxides with different hydrogen-bond situations. The satisfactory resolution and superior sensitivity of EDNMR as compared to the standard ENDOR (electron–nuclear double resonance) method are demonstrated.

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