The Temperature Dependence of Nitroxide Spin–Label Interaction Parameters: a High-Field EPR Study of Intramolecular Motional Contributions

High-field W-band electron paramagnetic resonance (EPR) spectroscopy was utilized to study the temperature dependence of the magnetic interaction parameters (g-, hyperfine-, quadrupole tensors) of two types of doublet-state nitroxide spin probes in glass-forming ortho-terphenyl solution: a five-membered ring system of pyrroline type (model for the commonly used methane thiosulfonate spin label) and a six-membered ring system of piperidine type (model for the commonly used TOAC spin label). The analysis of the g- and hyperfine tensors in terms of their isotropic and anisotropic parts reveals at least two mechanisms of motion that are responsible for the temperature dependence of the interaction parameters. The first mechanism is attributed to the overall small-angle motion of the nitroxide molecule in the glassy matrix; it leads to an averaging of the anisotropies of the EPR parameters. The second mechanism originates in an intramolecular out-of-plane motion of oxygen in the nitroxide group. This type of motion is evidenced by comparing the experimental findings for the spin-interaction parameters with the results of density functional theory calculations. The harmonic oxygen out-of-plane vibrations result in a variation of both the isotropic and anisotropic parts of the g- and hyperfine tensors. In contrast, the quadrupole tensor is not influenced by this vibration mechanism in the temperature range under study (90–240 K). Consequences of the applicability of such typical nitroxide radicals for probing details of their protein environment and for studying librational dynamics in frozen solutions are discussed.     

High-field EPR and site-directed spin labeling reveal a periodical polarity profile: The sequence 88 to 94 of the phototransducer NpHtrII in complex with sensory rhodopsin, NpSRII

Taking advantage of the improved spectral resolution of high-field electron paramagnetic resonance (EPR) at 95 GHz/3.4 T as compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the polarity profile in a protein-protein interface is obtained. Nitroxide spin label side chains are introduced at positions 88 to 94 in the AS-1 sequence of the membrane adjacent HAMP domain of the transducer protein, NpHtrII, which is reconstituted in complex with sensory rhodopsin, NpSRII fromNatronobacterium pharaonis. Position-dependent variations of the values of the nitroxide magnetic tensor componentsg _xx andA _zz suggest that the spin label side chains at positions 88 to 93 of AS-1 are located between a hydrophobic and a hydrophilic microenvironment. The observed periodicity of the polarity properties of the respective spin label microenvironment agrees with an α-helical secondary structure of this part of AS-1 and validates a recently published molecular model which locates residues 88 and 91 in the interface between helices F and G of NpSRII and AS-1 of NpHtrII close to the cytoplasmic lipid-water interface.     

Analysis of nitroxide spin label motion in a protein-protein complex using multiple frequency EPR spectroscopy

X- and W-band EPR spectra, at room and low temperatures, are reported for nitroxide spin labels attached to cysteine residues selectively introduced into two proteins, the DNase domain of colicin-E9 and its immunity protein, Im9. The dynamics of each site of attachment on the individual proteins and in the tight DNase-Im9 complex have been analysed by computer simulations of the spectra using a model of Brownian dynamics trajectories for the spin label and protein. Ordering potentials have been introduced to describe mobility of labels restricted by the protein domain. Label mobility varies with position from completely immobilised, to motionally restricted and to freely rotating. Bi-modal dynamics of the spin label have been observed for several sites. We show that W-band spectra are particularly useful for detection of anisotropy of spin label motion. On complex formation significant changes are observed in the dynamics of labels at the binding interface region. This work reveals multi-frequency EPR as a sensitive and valuable tool for detecting conformational changes in protein structure and dynamics especially in protein-protein complexes.     

Determination of absolute concentration of nitroxide radical by radio-frequency EPR imaging

The absolute concentrations of a nitroxide radical in samples in a loop-gap resonator (LGR) were determined by using a radio-frequency (about 720 MHz) electron paramagnetic resonance (EPR) imaging system. EPR imaging of phantoms containing a nitroxide radical, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (carbamoyl-PROXYL), dissolved in various concentrations of an aqueous sodium chloride solution was made to investigate the influence of dielectric losses and sample position within the LGR. As it was found that these influences on the signal intensity were sufficiently small (less than 6%), it is possible to use identical radical solutions in which the radical is dissolved in a known concentration as an internal marker. Two phantoms containing aqueous solutions of 3 mM (as a marker) and 1, 2, 3, 4, or 5 mM (as a sample) carbamoyl-PROXYL were placed together in the LGR. From EPR images of these phantoms, the absolute concentration of the sample could be calculated by using the gray-scale value (i.e., the signal intensity) of the marker and sample within a small margin of error (about 4%).     

Evaluation of spin labels for in-cell EPR by analysis of nitroxide reduction in cell extract of Xenopus laevis oocytes

Spin-label electron paramagnetic resonance (SL-EPR) spectroscopy has become a powerful and useful tool for studying structure and dynamics of biomacromolecules. However, utilizing these methods at physiological temperatures for in-cell studies is hampered by reduction of the nitroxide spin labels and thus short half-lives in the cellular environment. Consequently, reduction kinetics of two structurally different nitroxides was investigated in cell extracts of Xenopus laevis oocytes using rapid-scan cw-experiments at X-band. The five member heterocyclic ring nitroxide PCA (3-carboxy-2,2,5,5-tetramethylpyrrolidinyl-1-oxy) under investigation features much higher stability against intracellular reduction than the six member ring analog TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxilic acid) and is therefore a suitable spin label type for in-cell EPR. The kinetic data can be described according to the Michaelis–Menten model and thus suggest an enzymatic or enzyme-mediated reduction process.     

Biochemical EPR imaging of skin

EPR Imaging (EPRI) of spin labels is a powerful method for investigating skin and can give information about biochemical processes which are involved in numerous skin diseases. Furthermore it enables the non invasive investigation of the liberation, penetration and distribution of spin labelled drugs. The basis of these measurements is spectral spatial EPR imaging employing modulated field gradients and simultaneous field scans (MOSS). A skin region (?=6 mm) was treated with a 10 μl spin label solution (1 mM). EPR spectra of 128 points were recorded in 128 spatial planes resulting in a 128×128 image matrix. A spatial resolution of better than 10 μm can be obtained for a spectral line width of 0.1 mT and a gradient of 4 Tm^?1.In vivo imaging on mammalian skin can be performed by employing surface coils at S-band frequencies, 3 GHz.     

The aqueous reference for ESR oximetry

The interaction of molecular oxygen with derivatives of nitroxide EPR spin labels has been investigated using nuclear spin-relaxation spectroscopy in aqueous and nonaqueous solvents. The proton spin-lattice relaxation rate induced by oxygen provides a measure of the local concentration of oxygen, which we find is dependent on solvent. In water, the hydrophobic effect increases the local concentration of oxygen in the nonpolar portions of solute molecules. For nitroxides reduced to the hydroxylamine in aqueous solutions, we find that the local concentration of oxygen is approximately twice that associated with a free diffusion hard sphere limit, while in octane, this effect is absent. These results show that nitroxide based ESR oximetry may suffer a reference concentration shift of order a factor of two if the aqueous nitroxide spectrum or relaxation is used as the reference.     

A paramagnetic molecular voltmeter

We have developed a general electron paramagnetic resonance (EPR) method to measure electrostatic potential at spin labels on proteins to millivolt accuracy. Electrostatic potential is fundamental to energy-transducing proteins like myosin, because molecular energy storage and retrieval is primarily electrostatic. Quantitative analysis of protein electrostatics demands a site-specific spectroscopic method sensitive to millivolt changes. Previous electrostatic potential studies on macromolecules fell short in sensitivity, accuracy and/or specificity. Our approach uses fast-relaxing charged and neutral paramagnetic relaxation agents (PRAs) to increase nitroxide spin label relaxation rate solely through collisional spin exchange. These PRAs were calibrated in experiments on small nitroxides of known structure and charge to account for differences in their relaxation efficiency. Nitroxide longitudinal (R(1)) and transverse (R(2)) relaxation rates were separated by applying lineshape analysis to progressive saturation spectra. The ratio of measured R(1) increases for each pair of charged and neutral PRAs measures the shift in local PRA concentration due to electrostatic potential. Voltage at the spin label is then calculated using the Boltzmann equation. Measured voltages for two small charged nitroxides agree with Debye-Hückel calculations. Voltage for spin-labeled myosin fragment S1 also agrees with calculation based on the pK shift of the reacted cysteine.     

Overhauser dynamic nuclear polarization and molecular dynamics simulations using pyrroline and piperidine ring nitroxide radicals

The efficiency of Overhauser dynamic nuclear polarization (DNP) depends on the local dynamics modulating the dipolar coupling between the two interacting spins. By attaching nitroxide based spin labels to molecules and by measuring the ¹H DNP response of solvent water, information about the local hydration dynamics near the spin label can be obtained. However, there are two commonly used types of nitroxide ring structures; a pyrroline based and a piperidine based molecule. It is important to know when comparing different experiments, whether changes in DNP enhancements are due to changes in local hydration dynamics or because of the different spin label structures. In this study we investigate the key parameters affecting DNP signal enhancements for 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl, a 5-membered ring nitroxide radical, and for 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy, a 6-membered ring nitroxide radical. Using X-Band DNP, field cycling relaxometry, and molecular dynamics simulations, we conclude that the key parameters affecting the DNP amplitude of the ¹H signal of water to be equal when using either nitroxide. Thus, experiments measuring hydration dynamics using either type of spin labels may be compared.     

Investigating magnetically aligned phospholipid bilayers with EPR spectroscopy at 94 GHz

In this paper, we report our initial results on studying magnetically aligned phospholipid bilayers (bicelles) at high magnetic fields (approximately 3.4 T) with electron paramagnetic resonance (EPR) spectroscopy at 95 GHz (W-band). In order to characterize this system for W-band EPR studies, we have utilized the nitroxide spin probe 3beta-doxyl-5alpha-cholestane to demonstrate the effects of macroscopic bilayer alignment. At W-band due to the increase in magnetic field strength (when compared to X-band studies at 9.5 GHz) (S. M. Garber et al., J. Am. Chem. Soc. 121, 3240-3241 (1999)), we were able to examine magnetically aligned phospholipid bilayers at two orientations with the bilayer normal oriented either perpendicular or parallel (upon addition of YbCl3) with respect to the direction of the static magnetic field. Additionally, at a magnetic field of 3.4 T (g=2 resonance at W-band), we were able to study the parallel alignment with a lower concentration of Yb3+, thereby eliminating the possible unwanted effects associated with lanthanide-protein interactions and paramagnetic shifts and/or line broadening induced by the lanthanide ions. The development of this new spin label alignment technique will open up a whole new area of investigation for phospholipid bilayer systems and membrane protein EPR studies at high magnetic fields.     

Binding of MRI contrast agents to albumin: a high-field EPR study

High-field electron paramagnetic resonance (EPR) experiments to monitor binding of lipophilic Gd(III) complexes to human serum albumin (HSA) are described. It was observed that magnetic interactions between the nitroxide moiety ofn-doxyl-stearic acids bound to HSA and Gd(III) complexes resulted in broadening of nitroxide continuous-wave EPR spectra. The broadening effect can be well described by a one-parameter model of additional Lorentzian broadening At 95 GHz, continuous-wave EPR spectra from Gd(III) complexes are fully resolved from the nitroxide signal allowing for simultaneous and independent line shape analysis. Analysis of the line width broadening effects for spectra from a series ofn-doxyl-stearic acids bound to HSA indicated a progressive decrease of spin label-Gd(III) magnetic interactions along the fatty acid (FA) binding channel, consistent with binding of Gd-DOTAP complex in the vicinity of the main FA binding site. The substantial difference in spin label-metal interactions along the FA binding channel for lipophilic Gd(III) complexes with different chelates is indicative of binding to different sites. We also report measurements of dissociation constant for noncovalent binding of Gd(III) complexes to HSA on the basis of analyses of 95 GHz Gd(III) EPR line shapes.     

Self-Aggregation and Orientation of the Ion Channel-Forming Zervamicin IIA in the Membranes of ePC Vesicles Studied by cw EPR and ESEEM Spectroscopy

The ion channel-forming peptide antibiotic zervamicin A was studied in egg phosphocholin lipid membranes of large multilamellar vesicles (LMV) at 77 K. Continuous wave electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) methods combined with site-specific electron spin labeling were used to study the aggregation and immersion depth of two analog molecules, i.e., each monolabeled either at the N- or C-terminal end of the helical molecule. Analysis of the shape of the EPR spectra indicates that zervamicin molecules form aggregates in which the dipolar interaction between the spin labels at the N-terminus is substantially larger than that between the labels at the C-terminus. The ESEEM method was used to study the interaction between the nitroxide radical spin labels of the zervamicin molecules and deuterium nuclei in LMV, which were prepared using a D₂O buffer. It is established that the largest amplitude of deuterium modulation of the unpaired electron is observed for zervamicin molecules labeled at the N-terminus. Based on the analysis of the Fourier parameters of the deuterium modulated spectrum, a model of the immersion depth of the terminal ends of the zervamicin molecule in a lipid bilayer is formulated. All of the spin labels at the N-terminus are grouped at the lipid–water interface, whereas 60% of labels at the C-terminus are located at the lipid–water interface and 40% are more deeply inserted into the lipid bilayer.     

Permethyl-β-Cyclodextrin Spin-Labeled with Nitronyl Nitroxide: Synthesis and EPR Study

A method for synthesis of new covalently linked spin-labeled cyclodextrin (CD) via the attachment of nitronyl nitroxide 2-(4-hydroxyphenyl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl to permethylated β-cyclodextrin is described. Electron spin echo envelope modulation (ESEEM) studies demonstrate that the new spin-labeled CD exhibits dynamic equilibrium between conformations with radical fragment capping the cavity of CD and radical fragment located outside the cavity. In solution, nitronyl nitroxide attached to CD retains its sensitivity to nitric oxide (NO), as reaction with NO leads to formation of iminonitroxide fragment evidenced by continuous-wave (CW) electron paramagnetic resonance (EPR). At the same time, CW EPR study of the reaction with ascorbic acid shows that the described binding of nitronyl nitroxide to CD does not provide higher stability of radical towards the reduction, and the corresponding rate constants are close to those obtained for free nitronyl nitroxide. Plausible explanations of these observations are discussed.     

A loop resonator for slice-selective in vivo EPR imaging in rats

A loop resonator was developed for 300 MHz continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy and imaging in live rats. A single-turn loop (55 mm in diameter) was used to provide sufficient space for the rat body. Efficiency for generating a radiofrequency magnetic field of 38 microT/W(1/2) was achieved at the center of the loop. For the resonator itself, an unloaded quality factor of 430 was obtained. When a 350 g rat was placed in the resonator at the level of the lower abdomen, the quality factor decreased to 18. The sensitive volume in the loop was visualized with a bottle filled with an aqueous solution of the nitroxide spin probe 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy (3-CP). The resonator was shown to enable EPR imaging in live rats. Imaging was performed for 3-CP that had been infused intravenously into the rat and its distribution was visualized within the lower abdomen.     

Variable Coupling Scheme for High-Frequency Electron Spin Resonance Resonators Using Asymmetric Meshes

The sensitivity of a high-frequency electron spin resonance (ESR) spectrometer depends strongly on the structure used to couple the incident millimeter wave to the sample that generates the ESR signal. Subsequent coupling of the ESR signal to the detection arm of the spectrometer is also a crucial consideration for achieving high spectrometer sensitivity. In previous work, we found that a means for continuously varying the coupling was necessary for attaining high sensitivity reliably and reproducibly. We report here on a novel asymmetric mesh structure that achieves continuously variable coupling by rotating the mesh in its own plane about the millimeter-wave transmission-line optical axis. We quantify the performance of this device with nitroxide spin label spectra in both a lossy aqueous solution and a low-loss solid-state system. These two systems have very different coupling requirements and are representative of the range of coupling achievable with this technique. Lossy systems, in particular, are a demanding test of the achievable sensitivity and allow us to assess the suitability of this approach for applying high-frequency ESR, e.g., to the study of biological systems at physiological conditions. The variable coupling technique reported on here allows us to readily achieve a factor of ca. 7 improvement in the signal-to-noise ratio at 170 GHz and a factor of ca. 5 at 95 GHz over what has previously been reported for lossy samples.     

Molecular dynamics and spatial distribution of TOAC spin-labelled peptaibols studied in glassy liquid by echo-detected EPR spectroscopy

2,2,6,6-tetramethylpiperidine-l-oxyl-4-carboxylic acid (TOAC) spin-labelled analogues of the Aib-rich peptide (peptaibol) Trichogin GA IV are investigated in a glassy methanol/glycerol (70/30 v/v%) system, using conventional CW EPR and electron spin echo spectroscopy. Echo-detected (ED) EPR spectra indicate that the labels undergo restricted orientational motion (libration). Comparison with the small molecular spin probe Tempone shows that the dynamics of peptide molecules is determined by their structure and not by the surrounding medium. At the terminal positions (position 1 and 8) TOAC residues were found to be more flexible than at the central 4th position. Instantaneous diffusion mechanism in electron spin echo, which also contributes to the ED EPR spectra, was employed for deriving the local concentration of peptaibols. This approach may serve as a tool for investigation of peptide aggregation. In the studied model glassy solution the peptaibols were found to be randomly distributed.     

Polyelectrolyte Multilayers Studied by Electron Paramagnetic Resonance (EPR) Spin-Label Technique

A nitroxide spin label has been covalently attached to the polyelectrolyte poly(ethylene-alt-maleic acid) (P(E-alt-MA)) to study the interaction between this weak polyanion, the oppositely charged strong polycation poly(diallyldimethylammonium chloride) (PDADMAC) and water in swollen polyelectrolyte multilayers (PEM) by electron paramagnetic resonance (EPR) spectroscopy. If the spin-labeled polyanion has been used for the preparation of every double layer, the growth of the PEM film can be monitored by quantitative EPR. On the other hand, if the spin-labeled polyanion has been selectively placed in different layers in the PEM film the influence of the environment such as pH of the swelling medium of the mobility of the polyelectrolyte molecules positioned in the selected layer can be investigated.     

Intramolecular spin exchange in flexible short-chain biradicals in solutions with various viscosity

Intramolecular electron spin exchange as a function of temperature and solvent viscosity has been studied by X-band electron paramagnetic resonance (EPR) spectroscopy in two short-chain flexible nitroxide biradicals. Certain thermodynamic parameters of the conformational rearrangements were calculated from the EPR spectra. The process of spin exchange in these biradicals dissolved in six different alcohols is compared with that in a nonpolar solvent (toluene) and with the thermodynamic characteristics of the solvents. No correlation is found on a macroscopic level (solvent viscosity) but on a microscopic level (solvent longitudinal relaxation times) a distinct correlation is seen. The original results are compared with literature data.     

Conformational dynamics of some short-chain biradicals in solutions

Intramolecular electron spin exchange, as a function of temperature and the solvent nature, has been studied by X-band electron paramagnetic resonance (EPR) spectroscopy in five short-chain flexible nitroxide biradicals. Certain thermodynamic parameters of the conformational rearrangements were calculated from the EPR spectra. The process of spin exchange in short flexible biradicals has some peculiarities in comparison with that in long-chain molecules.