Structural, Biochemical, and Bioinformatic Basis for Identifying Radical SAM Cyclopropyl Synthases.

The importance of radical S-adenosyl-l-methionine (RS) enzymes in the maturation of ribosomally synthesized and post-translationally modified peptides (RiPPs) continues to expand, specifically for the RS-SPASM subfamily. We recently discovered an RS-SPASM enzyme that installs a carbon-carbon bond between the geminal methyls of valine residues, resulting in the formation of cyclopropylglycine (CPG). Here, we sought to define the family of cyclopropyl (CP) synthases because of the importance of cyclopropane scaffolds in pharmaceutical development. Using RadicalSAM.org, we bioinformatically expanded the family of CP synthases and assigned unique peptide sequences to each subclade. We identified a unique RiPP biosynthetic pathway that encodes a precursor peptide, TigB, with a repeating TIGSVS motif. Using LCMS and NMR techniques, we show that the RS enzyme associated with the pathway, TigE, catalyzes the formation of a methyl-CPG from the conserved isoleucine residing in the repeating motif of TigB. Furthermore, we obtained a crystal structure of TigE, which reveals an unusual tyrosyl ligation to the auxiliary I [4Fe-4S] cluster, provided by a glycine-tyrosine-tryptophan motif unique to all CP synthases. Further, we show that this unique tyrosyl ligation is absolutely required for TigE activity. Together, our results provide insight into how CP synthases perform this unique reaction.

Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 × 106 M-1, is at least 1 order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase in the rotational correlation time, τrot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τrot and electron spin coherence time, Tm, along with the narrowing of interspin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4-9, compared to the common spin label such as MTSL, which is not affected by CB-7. Interspin distances of 3 nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating an undisturbed structure and conformation of the protein.

Electron Paramagnetic Resonance, Electronic Ground State, and Electron Spin Relaxation of Seven Lanthanide Ions Bound to Lanmodulin and the Bioinspired Chelator, 3,4,3-LI(1,2-HOPO).

The electron paramagnetic resonance (EPR) spectra of lanthanide(III) ions besides Gd3+ , bound to small-molecule and protein chelators, are uncharacterized. Here, the EPR properties of 7 lanthanide(III) ions bound to the natural lanthanide-binding protein, lanmodulin (LanM), and the synthetic small-molecule chelator, 3,4,3-LI(1,2-HOPO) ("HOPO"), were systematically investigated. Echo-detected pulsed EPR spectra reveal intense signals from ions for which the normal continuous-wave first-derivative spectra are negligibly different from zero. Spectra of Kramers lanthanide ions Ce3+ , Nd3+ , Sm3+ , Er3+ , and Yb3+ , and non-Kramers Tb3+ and Tm3+ , bound to LanM are more similar to the ions in dilute aqueous:ethanol solution than to those coordinated with HOPO. Lanmodulins from two bacteria, with distinct metal-binding sites, had similar spectra for Tb3+ but different spectra for Nd3+ . Spin echo dephasing rates (1/Tm ) are faster for lanthanides than for most transition metals and limited detection of echoes to temperatures below ~6 to 12 K. Dephasing rates were environment dependent and decreased in the order water:ethanol>LanM>HOPO, which is attributed to decreasing librational motion. These results demonstrate that the EPR spectra and relaxation times of lanthanide(III) ions are sensitive to coordination environment, motivating wider application of these methods for characterization of both small-molecule and biomolecule interactions with lanthanides.

Imaging Reactive Oxygen Radicals in Excised Mouse Lung Trapped by Reaction with Hydroxylamine Probes Using 1 GHz Rapid Scan Electron Paramagnetic Resonance.

PURPOSE: Oxidative stress is proposed to be critical in acute lung disease, but methods to monitor radicals in lungs are lacking. Our goal is to develop low-frequency electron paramagnetic resonance (EPR) methods to monitor radicals that contribute to the disease. PROCEDURES: Free radicals generated in a lipopolysaccharide-induced mouse model of acute respiratory distress syndrome reacted with cyclic hydroxylamines CPH (1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride) and DCP-AM-H (4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid), which were converted into the corresponding nitroxide radicals, CP• and DCP•. The EPR signals of the nitroxide radicals in excised lungs were imaged with a 1 GHz EPR spectrometer/imager that employs rapid scan technology. RESULTS: The small numbers of nitroxides formed by reaction of the hydroxylamine with superoxide result in low signal-to-noise in the spectra and images. However, since the spectral properties of the nitroxides are known, we can use prior knowledge of the line shape and hyperfine splitting to fit the noisy data, yielding well-defined spectra and images. Two-dimensional spectral-spatial images are shown for lung samples containing (4.5 ± 0.5) ×1014 CP• and (9.9 ± 1.0) ×1014 DCP• nitroxide spins. These results suggest that a probe that accumulates in cells gives a stronger nitroxide signal than a probe that is more easily washed out of cells. CONCLUSION: The nitroxide radicals in excised mouse lungs formed by reaction with hydroxylamine probes CPH and DCP-AM-H can be imaged at 1 GHz.

Cyclic Disulfide-Bridged Dinitroxide Biradical for Measuring Thiol Redox Status by Electron Paramagnetic Resonance.

Among low-molecular-weight thiols, glutathione (GSH) is the main antioxidant in the cell, and its concentration is an indicator of the redox status. A cyclic disulfide-linked dinitroxide was designed for monitoring GSH by electron-paramagnetic resonance (EPR) spectroscopy. Reaction of the disulfide with GSH and three other thiols was measured at 9.6 GHz (X-band) and shown to be of first order in thiols. It is proposed that the reaction of the disulfide with 1 equiv of thiolate produced a short-lived intermediate that reacts with 1 equiv of thiolate to produce the cleavage product. The equilibrium ratio of the cleaved and intact disulfide is a measure of the redox state. Since the long-term goal is to use the disulfide to probe physiology in vivo, the feasibility of EPR spectroscopy and imaging of the disulfide and its cleavage product was demonstrated at 1 GHz (L-band).

Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 × 106 M-1, is at least one order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase of the rotational correlation time, τrot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τrot and electron spin coherence time, Tm, along with the narrowing of inter-spin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4 - 9, compared to the common spin label such as MTSL, which is not affected by CB-7. Inter-spin distances of 3-nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating undisturbed structure and conformation of the protein.

Electron spin echo spectra and characterization of electronic ground states for lanthanide ions in acidic water:ethanol glasses.

X-band EPR absorption spectra were obtained by field-swept echo-detection for 10 mM Ce3+, Nd3+, Sm3+, Tb3+, Er3+, Tm3+, and Yb3+ in glassy acidic 1:1 water:ethanol at 4.2 to 6 K. The slowly-varying signals extend over thousands of gauss and are more readily detected by spin echo than by continuous wave EPR. For lanthanide ions spin-orbit coupling is strong and J is a good quantum number. The microwave power required to achieve a π/2 pulse depends on quantum numbers J and mJ and the gJ for the levels involved in the EPR transition, so it can be used to characterize the electronic ground state. For Nd3+, Sm3+, Er3+, and Tm3+ the power required for a π/2 pulse is consistent with values calculated for the expected value of gJ and transitions involving mJ = ±1/2 or mJ = ±1,0. However for Ce3+, Gd3+, Tb3+, and Yb3+ the microwave power required for a π/2 pulse is larger than predicted. For Ce3+, Tb3+, and Yb3+ it is proposed that mixing of levels results in contributions to the transitions from higher values of mJ. For Gd3+ the discrepancy is attributed to overlapping transitions with varying values of mJ.

Direct Measurement and Imaging of Redox Status with Electron Paramagnetic Resonance.

Significance: Fundamental to the application of tissue redox status to human health is the quantification and localization of tissue redox abnormalities and oxidative stress and their correlation with the severity and local extent of disease to inform therapy. The centrality of the low-molecular-weight thiol, glutathione, in physiological redox balance has long been appreciated, but direct measurement of tissue thiol status in vivo has not been possible hitherto. Recent advances in instrumentation and molecular probes suggest the feasibility of real-time redox assessment in humans. Recent Advances: Recent studies have demonstrated the feasibility of using low-frequency electron paramagnetic resonance (EPR) techniques for quantitative imaging of redox status in mammalian tissues in vivo. Rapid-scan (RS) EPR spectroscopy and imaging, new disulfide-dinitroxide spin probes, and novel analytic techniques have led to significant advances in direct, quantitative imaging of thiol redox status. Critical Issues: While novel RS EPR imaging coupled with first-generation molecular probes has demonstrated the feasibility of imaging thiol redox status in vivo, further technical advancements are desirable and ongoing. These include developing spin probes that are tailored for specific tissues with response kinetics tuned to the physiological environment. Equally critical are RS instrumentation with higher signal-to-noise ratio and minimal signal distortion, as well as optimized imaging protocols for image acquisition with sparsity adapted to image information content. Future Directions: Quantitative images of tissue glutathione promise to enable acquisition of a general image of mammalian and potentially human tissue health.

Synthesis and characterization of a biocompatible 13C1 isotopologue of trityl radical OX071 for in vivo EPR viscometry.

We describe the synthesis, characterization, and application of an isotopologue of the trityl radical OX071, labeled with 13C at the central carbon (13C1). This spin probe features large anisotropy of the hyperfine coupling with the 13C1 (I = 1/2), leading to an EPR spectrum highly sensitive to molecular tumbling. The high biocompatibility and lack of interaction with blood albumin allow for systemic delivery and in vivo measurement of tissue microviscosity by EPR.

Impact of Chlorine Substitution on Electron Spin Relaxation of a Trityl Radical.

A perchlorotriarylmethyl tricarboxylic acid radical 99% enriched in 13C at the central carbon (13C1-PTMTC) was characterized in phosphate buffered saline solution (pH = 7.2) (PBS) at ambient temperature. Samples immobilized in 1:1 PBS:glycerol or in 9:1 trehalose:sucrose were studied as a function of temperature. Isotope enrichment at C1 creates a trityl that can be used to accurately measure microscopic viscosity. Understanding of the impact of the 13C hyperfine interaction on electron spin relaxation is important for application of this trityl in oximetry and distance measurements. The anisotropic 13C1 hyperfine couplings (Ax = Ay = 24 ± 2 MHz, Az = 200 ± 1 MHz) are larger than for the related 13C1-perdeuterated Finland trityl (13C1-dFT) and the g anisotropy (gx = 2.0013, gy = 2.0016, gz = 2.0042) is slightly larger than for 13C1-dFT. The tumbling correlation times (τR) for 13C1-PTMTC are 0.20 ± 0.02 ns in PBS and 0.40 ± 0.05 ns in 3:1 PBS:glycerol, which are shorter than for 13C1-dFT in the same solutions. T1 for 13C1-PTMTC is 3.5 ± 0.5 µs in PBS and 5.3 ± 0.4 µs in 3:1 PBS:glycerol, which are shorter than for 13C1-dFT due to faster tumbling, larger anisotropy of the 13C1 hyperfine, and about 30% larger contribution from the local mode. In immobilized samples T1 for 13C1-PTMTC is similar to that for 13C1-dFT and other trityls without chlorine or 13C1 substituents, indicating that the 13C1 and Cl substituents on the phenyl rings have little impact on T1. The temperature dependence of T1 was modeled with contributions from the direct, Raman, and local mode processes. Broadening of CW linewidths of about 0.6 G in fluid solution and about 2 G in rigid lattice is attributed to unresolved 35,37Cl hyperfine couplings.

Advances in rapid scan EPR spectroscopy.

Progress has been made in hardware for low frequencies, demonstrations of rapid frequency scans, hybrid instrumentation, and improved deconvolution software. The recent availability of the commercial Bruker BioSpin rapid scan accessory for their X-band EMX and Elexsys systems makes this technique available to a wide range of users without the need to construct their own system. Developments at lower frequencies are underway in several labs with the goal of facilitating in vivo and preclinical rapid scan imaging. Development of new deconvolution algorithms will make data processing more robust. Frequency scans have substantial promise at higher frequencies. New examples of applications show the wide applicability and advantages of rapid scan.

Identification of a poly-cyclopropylglycine-containing peptide via bioinformatic mapping of radical S-adenosylmethionine enzymes.

Peptide-derived natural products are a large class of bioactive molecules that often contain chemically challenging modifications. In the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs), radical-SAM (rSAM) enzymes have been shown to catalyze the formation of ether, thioether, and carbon-carbon bonds on the precursor peptide. The installation of these bonds typically establishes the skeleton of the mature RiPP. To facilitate the search for unexplored rSAM-dependent RiPPs for the community, we employed a bioinformatic strategy to screen a subfamily of peptide-modifying rSAM enzymes which are known to bind up to three [4Fe-4S] clusters. A sequence similarity network was used to partition related families of rSAM enzymes into >250 clusters. Using representative sequences, genome neighborhood diagrams were generated using the Genome Neighborhood Tool. Manual inspection of bacterial genomes yielded numerous putative rSAM-dependent RiPP pathways with unique features. From this analysis, we identified and experimentally characterized the rSAM enzyme, TvgB, from the tvg gene cluster from Halomonas anticariensis. In the tvg gene cluster, the precursor peptide, TvgA, is comprised of a repeating TVGG motif. Structural characterization of the TvgB product revealed the repeated formation of cyclopropylglycine, where a new bond is formed between the γ-carbons on the precursor valine. This novel RiPP modification broadens the functional potential of rSAM enzymes and validates the proposed bioinformatic approach as a practical broad search tool for the discovery of new RiPP topologies.

Electron paramagnetic resonance characterization and electron spin relaxation of manganate ion in glassy alkaline LiCl solution and doped into Cs2SO4.

Manganate ion, MnO42-, has important roles in catalysis and potential roles in water treatment. EPR spectra of MnO42- in a glassy alkaline solution of concentrated LiCl at X-band and Q-band at 80 K exhibit g1 = 1.9776 ± 0.001, g2 = 1.9677 ± 0.001, g3 = 1.9560 ± 0.001 and A1 = 182 ± 9, A2 = 275 ± 15, and A3 = 400 ± 15 MHz. In Cs2SO4 the spectra were simulated with 1.908 ± 0.001, g2 = 1.909 ± 0.001, g3 = 1.937 ± 0.001 and A1 = 90 ± 20, A2 = 100 ± 20, and A3 = 400 ± 15 MHz. Simulations required large distributions in A values which suggests that hyperfine splittings are sensitive to differences in geometry. Continuous wave spectra are observable at 80 K in glassy alkaline LiCl, but only up to about 20 K in Cs2SO4. In glassy alkaline LiCl electron spin relaxation was measured at X-band using spin echo and inversion recovery from 4.2 to 60 K. Tm is 4.6 µs at 4.2 K and decreases at higher temperatures as it becomes driven by T1. T1 decreases from ca. 34 ms at 4.2 K to ca. 240 ns at 60 K. Tm and T1 in Cs2SO4 are too short to measure by electron spin echo. The distorted tetrahedral geometry of MnO42- results in faster relaxation than for other 3d1 spin systems that have square pyramidal (C4v) or distorted octahedral geometries.

Spin-spin interaction and relaxation in two trityl-nitroxide diradicals.

Trityl-nitroxides show substantial promise as polarizing agents in solid state dynamic nuclear polarization. To optimize performance it is important to understand the impact of spin-spin interactions on relaxation times of the diradicals. CW spectra and electron spin relaxation were measured for two trityl-nitroxides that differ in the substituents on the amide linker and have different strengths of the exchange interaction J. Analysis of the EPR spectra in terms of overlapping AB spin-spin splitting patterns explains the impact of J on various regions of the spectra. Even modest values of J are large relative to the separation between trityl and nitroxide resonances for some nitrogen nuclear spin state. Two conformations for each diradical were observed in CW spectra in fluid solution at X-band and Q-band. For one diradical J = 15 G (83%) and 5 G (17%) at 293 K, and J = 27 G (67%) and 3 G (33%) with interspin distances of 16 Å and 12 Å, respectively, at 80 K. For the second diradical the exchange interaction is stronger: the two conformations in fluid solution at 293 K had J = 113 G (67%) and 59 G (33%) and at 80 K the value of J was 43 G and there were two conformations with interspin distances of 13 and 11.5 Å. The observation of two conformations for each diradical, with different values of J, demonstrates the dependence of their exchange interactions on through-bond orbital interactions. X-band values of spin relaxation rates 1/T1 and 1/Tm at 80 to 120 K for the trityl-nitroxides are similar to values for nitroxide mono-radicals, and faster than for trityl radicals. These observations show that even for a relatively small value of J, the nitroxide is very effective in enhancing the relaxation of the more slowly relaxing trityl.

Bis-Spiro-Oxetane and Bis-Spiro-Tetrahydrofuran Pyrroline Nitroxide Radicals: Synthesis and Electron Spin Relaxation Studies.

Synthesis of bis-spiro-oxetane and bis-spiro-tetrahydrofuran pyrroline nitroxide radicals relies on the Mitsunobu reaction-mediated double cyclizations of N-Boc protected pyrroline tetraols. Structures of the nitroxide radicals are supported by X-ray crystallography. In a trehalose/sucrose matrix at room temperature, the bis-spiro-oxetane nitroxide radical possesses electron spin coherence time, Tm ≈ 0.7 µs. The observed enhanced Tm is most likely associated with strong hydrogen bonding of oxetane moieties to the trehalose/sucrose matrix.

Perchlorinated Triarylmethyl Radical 99% Enriched 13C at the Central Carbon as EPR Spin Probe Highly Sensitive to Molecular Tumbling.

Soluble stable radicals are used as spin probes and spin labels for in vitro and in vivo electron paramagnetic resonance (EPR) spectroscopy and imaging applications. We report the synthesis and characterization of a perchlorinated triarylmethyl radical enriched 99% at the central carbon, 13C1-PTMTC. The anisotropy of the hyperfine splitting with the 13C1 (Ax = 26, Ay = 25, Az = 199.5 MHz) and the g (gx = 2.0015, gy = 2.0015, gz = 2.0040) are responsible for a strong effect of the radical tumbling rate on the EPR spectrum. The rotational correlation time can be determined by spectral simulation or via the line width or the apparent Az after calibration, so the spin probe 13C1-PTMTC can be used to measure media microviscosity with high sensitivity.

Electron paramagnetic resonance of lanthanides.

Many applications of lanthanides exploit their electron spin relaxation properties. Double electron-electron measurements of distances are possible because of the relatively long relaxation times of Gd3+. Relaxation enhancement measurements of distance are possible because of the much shorter relaxation times of other lanthanides. Magnetic resonance imaging contrast agents use the long relaxation time of the S-state Gd3+ ion, and NMR shift reagents use the fast relaxation of selected other lanthanides. Other than Gd3+ and the isoelectronic Eu2+ ion, spin relaxation of the lanthanides is so fast that their EPR spectra can be observed only in the liquid helium temperature range. In this chapter the EPR properties of each of the lanthanides is briefly summarized, with an emphasis on electron spin relaxation.

Ligand control of low-frequency electron paramagnetic resonance linewidth in Cr(III) complexes.

Understanding how the ligand shell controls low-frequency electron paramagnetic resonance (EPR) spectroscopic properties of metal ions is essential if they are to be used in EPR-based bioimaging schemes. In this work, we probe how specific variations in the ligand structure impact L-band (ca. 1.3 GHz) EPR spectroscopic linewidths in the trichloride salts of five Cr(iii) complexes: [Cr(RR-dphen)3]3+ (RR-dphen = (1R,2R)-(+)-diphenylethylenediamine, 1), [Cr(en)3]3+ (en = ethylenediamine, 2), [Cr(me-en)3]3+ (me-en = 1,2-diaminopropane, 3), [Cr(tn)3]3+ (tn = 1,3-diaminopropane, 4) [Cr(trans-chxn)3]3+ (trans-chxn = trans-(±)-1,2-diaminocyclohexane, 5). Spectral broadening varies in a nonintuitive manner across the series, showing the sharpest peaks for 1 and broadest for 5. Molecular dynamics simulations provide evidence that the broadening is correlated to rigidity in the inner coordination sphere and reflected in ligand-dependent distribution of Cr-N bond distances that can be found in frozen solution.

Trehalose as an alternative to glycerol as a glassing agent for in vivo DNP MRI.

PURPOSE: In dynamic nuclear polarization (DNP), the solution needs to form a glass to attain significant levels of polarization in reasonable time periods. Molecules that do not form glasses by themselves are often mixed with glass forming excipients. Although glassing agents are often essential in DNP studies, they have the potential to perturb the metabolic measurements that are being studied. Glycerol, the glassing agent of choice for in vivo DNP studies, is effective in reducing ice crystal formation during freezing, but is rapidly metabolized, potentially altering the redox and adenosine triphosphate balance of the system. METHODS: DNP buildup curves of 13 C urea and alanine with OX063 in the presence of trehalose, glycerol, and other polyol excipients were measured as a function of concentration. T1 and Tm relaxation times for OX063 in the presence of trehalose were measured by EPR. RESULTS: Approximately 15-20 wt% trehalose gives a glass that polarizes samples more rapidly than the commonly used 60%-wt formulation of glycerol and yields similar polarization levels within clinically relevant timeframes. CONCLUSIONS: Trehalose may be an attractive biologically inert alternative to glycerol for situations where there may be concerns about glycerol's glucogenic potential and possible alteration of the adenosine triphosphate/adenosine diphosphate and redox balance.

Rapid-Scan Electron Paramagnetic Resonance of Highly Resolved Hyperfine Lines in Organic Radicals.

X-band (ca. 9 GHz) fluid solution rapid-scan electron paramagnetic resonance spectra are reported for radicals with multiline spectra and resolution of hyperfine lines as narrow as 30 mG. Highly-resolved spectra of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy, diphenylnitroxide, galvinoxyl, and perylene cation radical with excellent signal-to-noise are shown, demonstrating the capabilities of the rapid-scan technique to characterize very small, well-resolved hyperfine couplings. To acquire high resolution spectra the signal bandwidth must be less than the resonator bandwidth. Signal bandwidth is inversely proportional to linewidth and proportional to scan rate. Resonator bandwidth is inversely proportional to resonator Q. Proper selection of scan rate and resonator Q is needed to achieve resolution of closely-spaced narrow EPR lines.