A radical approach to radicals

Nitroxide radicals are characterized by a long-lived spin-unpaired electronic ground state and are strongly sensitive to their chemical surroundings. Combined with electron paramagnetic resonance spectroscopy, these electronic features have led to the widespread application of nitroxide derivatives as spin labels for use in studying protein structure and dynamics. Site-directed spin labelling requires the incorporation of nitroxides into the protein structure, leading to a new protein–ligand molecular model. However, in protein crystallographic refinement nitroxides are highly unusual molecules with an atypical chemical composition. Because macromolecular crystallography is almost entirely agnostic to chemical radicals, their structural information is generally less accurate or even erroneous. In this work, proteins that contain an example of a radical compound (Chemical Component Dictionary ID MTN) from the nitroxide family were re-refined by defining its ideal structural parameters based on quantum-chemical calculations. The refinement results show that this procedure improves the MTN ligand geometries, while at the same time retaining higher agreement with experimental data.

In situ distance measurements in a membrane transporter using maleimide functionalized orthogonal spin labels and 5-pulse electron double resonance spectroscopy

Spectroscopic investigation of membrane proteins in their native environment is a challenging task. Earlier we demonstrated the feasibility to measure precise distances within outer membrane proteins in E. coli and native membranes using methanethiosulfonate (MTS) functionalized labels combined with pulsed electron double resonance spectroscopy. Here we show the application of maleimide functionalized Gd(III), nitroxide, and trityl labels for in situ distance measurement using the cobalamin transporter BtuB. These labels enabled distance measurements for BtuB in E. coli and native outer membranes and in the membranes maleimide-Gd-DOTA also is effective. Further, we show that the observable dipolar evolution time can be significantly prolonged in the native environments using the Carr-Purcell 5-pulse electron double resonance sequence. For a nitroxide-nitroxide pair, application of sech/tanh inversion pulses substantially suppressed the 4-pulse artifact at the Q- band frequency. In the case of a nitroxide-trityl pair, Gaussian pump pulses of varying amplitude are sufficient to suppress the artifact to the typical noise level. The feasibility of a range of bioresistant spin labels and the 5-pulse electron double resonance offers promising tools for investigating heterooligomeric membrane protein complexes in their native environment.

4-Dialkylamino-2,5-dihydroimidazol-1-oxyls with Functional Groups at the Position 2 and at the Exocyclic Nitrogen: The pH-Sensitive Spin Labels

Local acidity and electrostatic interactions are associated both with catalytic properties and the adsorption activity of various materials, and with the vital functions of biomolecules. The observation of acid–base equilibria in stable free radicals using EPR spectroscopy represents a convenient method for monitoring pH changes and the investigation of surface electrostatics, the advantages of which are especially evident in opaque and turbid samples and in porous materials such as xerogels. Imidazoline nitroxides are the most commonly used pH-sensitive spin probes and labels due to the high sensitivity of the parameters of the EPR spectra to pH changes, their small size, and their well-developed chemistry. In this work, several new derivatives of 4-(N,N-dialkylamino)-2,5-dihydrioimidazol-1-oxyl, with functional groups suitable for specific binding, were synthesized. The dependence of the parameters of their EPR spectra on pH was studied. Several showed a pKa close to 7.4, following the pH changes in a normal physiological range, and some demonstrated a monotonous change of the hyperfine coupling constant by 0.14 mT upon pH variation by four units.

A Comparison of Cysteine-Conjugated Nitroxide Spin Labels for Pulse Dipolar EPR Spectroscopy

The structure-function and materials paradigms drive research on the understanding of structures and structural heterogeneity of molecules and solids from materials science to structural biology. Functional insights into complex architectures are often gained from a suite of complementary physicochemical methods. In the context of biomacromolecular structures, the use of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) has become increasingly popular. The main interest in PDS is providing long-range nanometre distance distributions that allow for identifying macromolecular topologies, validating structural models and conformational transitions as well as docking of quaternary complexes. Most commonly, cysteines are introduced into protein structures by site-directed mutagenesis and modified site-specifically to a spin-labelled side-chain such as a stable nitroxide radical. In this contribution, we investigate labelling by four different commercial labelling agents that react through different sulfur-specific reactions. Further, the distance distributions obtained are between spin-bearing moieties and need to be related to the protein structure via modelling approaches. Here, we compare two different approaches to modelling these distributions for all four side-chains. The results indicate that there are significant differences in the optimum labelling procedure. All four spin-labels show differences in the ease of labelling and purification. Further challenges arise from the different tether lengths and rotamers of spin-labelled side-chains; both influence the modelling and translation into structures. Our comparison indicates that the spin-label with the shortest tether in the spin-labelled side-group, (bis-(2,2,5,5-Tetramethyl-3-imidazoline-1-oxyl-4-yl) disulfide, may be underappreciated and could increase the resolution of structural studies by PDS if labelling conditions are optimised accordingly.

Assessment of Aspartate and Bicarbonate Produced From Hyperpolarized [1-13C]Pyruvate as Markers of Renal Gluconeogenesis

As both a consumer and producer of glucose, the kidney plays a significant role in glucose homeostasis. Measuring renal gluconeogenesis requires invasive techniques, and less invasive methods would allow renal gluconeogenesis to be measured more routinely. Magnetic resonance spectroscopy and imaging of infused substrates bearing hyperpolarized carbon-13 spin labels allows metabolism to be detected within the body with excellent sensitivity. Conversion of hyperpolarized 1-13C pyruvate in the fasted rat liver is associated with gluconeogenic flux through phosphoenolpyruvate carboxykinase (PEPCK) rather than pyruvate dehydrogenase (PDH), and this study tested whether this was also the case in the kidney. The left kidney was scanned in fed and overnight-fasted rats either with or without prior treatment by the PEPCK inhibitor 3-mercaptopicolinic acid (3-MPA) following infusion of hyperpolarized 1-13C pyruvate. The 13C-bicarbonate signal normalized to the total metabolite signal was 3.2-fold lower in fasted rats (p = 0.00073) and was not significantly affected by 3-MPA treatment in either nutritional state. By contrast, the normalized [1-13C]aspartate signal was on average 2.2-fold higher in the fasted state (p = 0.038), and following 3-MPA treatment it was 2.8-fold lower in fed rats and 15-fold lower in fasted rats (p = 0.001). These results confirm that, unlike in the liver, most of the pyruvate-to-bicarbonate conversion in the fasted kidney results from PDH flux. The higher conversion to aspartate in fasted kidney and the marked drop following PEPCK inhibition demonstrate the potential of this metabolite as a marker of renal gluconeogenesis.

Hydrogen-Bonded Complexes of Neutral Nitroxide Radicals with 2-Propanol Studied by Multifrequency EPR/ENDOR

The hydrogen bond plays a key role in weak directional intermolecular interactions. It is operative in determining molecular conformation and aggregation, and controls the function of many chemical systems, ranging from inorganic, organic to biological molecules. Although an enormous amount of spectroscopic information has been collected about hydrogen-bond formation between molecules with closed-shell electronic configuration, the details of such interactions between open-shell radicals and closed-shell molecules are still rare. Here we report on an investigation of hydrogen-bonded complexes between pyrroline-type as well as piperidine-type neutral nitroxide radicals and an alcohol, i.e., 2-propanol. These nitroxide radicals are commonly used as EPR spin labels and probes. To obtain information on the geometry of the complexes and their electronic structure, multi-resonance EPR techniques at various microwave frequencies (X-, Q-, W-band, 244 GHz) have been employed in conjunction with DFT calculations. The planar five-membered ring system of the pyrroline-type nitroxide radical was found to form exclusively well-defined in-plane σ-type hydrogen-bonded complexes with one 2-propanol molecule in the first solvation shell in frozen solution. The measured hyperfine parameters of the hydrogen-bridge proton and the internal magnetic parameters describing the electron Zeeman and the electron-nuclear hyperfine and nuclear quadrupole interactions are in good agreement with values predicted by state-of-the-art DFT calculations. In contrast, multi-resonance EPR on the non-planar six-membered ring system of the piperidine-type nitroxide radical (TEMPOL) reveals a more complex situation, i.e., a mixture of a σ-type with, presumably, an out-of-plane π-type complex, both present in comparable fraction in frozen solution. For TEMPOL, the DFT calculations failed to predict magnetic interaction parameters that are in good agreement with experiment, apparently due to the considerable flexibility of the nitroxide and hydrogen-bonded complex. The detailed information about nitroxide/solvent complexes is of particular importance for Dynamic Nuclear Polarization (DNP) and site-directed spin-labeling EPR studies that employ nitroxides as polarizing agents or spin labels, respectively.

A Computational Journey across Nitroxide Radicals: From Structure to Spectroscopic Properties and Beyond

Nitroxide radicals are characterized by a long-lived open-shell electronic ground state and are strongly sensitive to the chemical environment, thus representing ideal spin probes and spin labels for paramagnetic biomolecules and materials. However, the interpretation of spectroscopic parameters in structural and dynamic terms requires the aid of accurate quantum chemical computations. In this paper we validate a computational model rooted into double-hybrid functionals and second order vibrational perturbation theory. Then, we provide reference quantum chemical results for the structures, vibrational frequencies and other spectroscopic features of a large panel of nitroxides of current biological and/or technological interest.

Electron Spin Resonance Evaluation of Buccal Membrane Fluidity Alterations by Sodium Caprylate and L-Menthol

The ability of sodium caprylate and l-menthol to fluidize phospholipid bilayers composed of lipids simulating the buccal epithelium was investigated using electron spin resonance (ESR) to evaluate the action of these agents as permeation enhancers. 5-Doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA) were used as spin labels to identify alterations in membrane fluidity near the polar head groups or inner acyl regions of the lipid bilayer, respectively. The molecular motion of both 5-DSA and 16-DSA showed increased disorder near the polar and inner hydrophobic regions of the bilayer in the presence of sodium caprylate suggesting fluidization in both the regions, which contributes to its permeation enhancing effects. L-menthol decreased the order parameter for 16-DSA, showing membrane fluidization only in the inner acyl regions of the bilayer, which also corresponded to its weaker permeation enhancing effects. The rapid evaluation of changes in fluidity of the bilayer in the presence of potential permeation enhancers using ESR enables improved selection of effective permeation enhancers and enhancer combinations based on their effect on membrane fluidization.

A Simple Method of Synthesis of 3-Carboxy-2,2,5,5-Tetraethylpyrrolidine-1-oxyl and Preparation of Reduction-Resistant Spin Labels and Probes of Pyrrolidine Series

Stable free radicals are widely used as molecular probes and labels in various biophysical and biomedical research applications of magnetic resonance spectroscopy and imaging. Among these radicals, sterically shielded nitroxides of pyrrolidine series demonstrate the highest stability in biological systems. Here, we suggest new convenient procedure for preparation of 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl, a reduction-resistant analog of widely used carboxy-Proxyl, from cheap commercially available reagents with the yield exceeding the most optimistic literature data. Several new spin labels and probes of 2,2,5,5-tetraethylpyrrolidine-1-oxyl series were prepared and reduction of these radicals in ascorbate solutions, mice blood and tissue homogenates was studied.

Protein functional dynamics from the rigorous global analysis of DEER data: Conditions, components, and conformations

The potential of spin labeling to reveal the dynamic dimension of macromolecules has been recognized since the dawn of the methodology in the 1960s. However, it was the development of pulsed electron paramagnetic resonance spectroscopy to detect dipolar coupling between spin labels and the availability of turnkey instrumentation in the 21st century that realized the full promise of spin labeling. Double electron-electron resonance (DEER) spectroscopy has seen widespread applications to channels, transporters, and receptors. In these studies, distance distributions between pairs of spin labels obtained under different biochemical conditions report the conformational states of macromolecules, illuminating the key movements underlying biological function. These experimental studies have spurred the development of methods for the rigorous analysis of DEER spectroscopic data along with methods for integrating these distributions into structural models. In this tutorial, we describe a model-based approach to obtaining a minimum set of components of the distance distribution that correspond to functionally relevant protein conformations with a set of fractional amplitudes that define the equilibrium between these conformations. Importantly, we review and elaborate on the error analysis reflecting the uncertainty in the various parameters, a critical step in rigorous structural interpretation of the spectroscopic data.