Simulation and design of shaped pulses beyond the piecewise-constant approximation

Response functions of resonant circuits create ringing artefacts if their input changes rapidly. When physical limits of electromagnetic spectroscopies are explored, this creates two types of problems. Firstly, simulation: the system must be propagated accurately through every response transient, this may be computationally expensive. Secondly, optimal control: circuit response must be taken into account; it may be advantageous to design pulses that are resilient to such distortions. At the root of both problems is the popular piecewise-constant approximation for control sequences in the rotating frame; in magnetic resonance it has persisted since the earliest days and has become entrenched in the commercially available hardware. In this paper, we report an implementation and benchmarks of recent Lie-group methods that can efficiently simulate and optimise smooth control sequences

A Chemically Competent Thiosulfuranyl Radical on the Escherichia coli Class III Ribonucleotide Reductase

The class III ribonucleotide reductases (RNRs) are glycyl radical (G•) enzymes that provide the balanced pool of deoxynucleotides required for DNA synthesis and repair in many facultative and obligate anaerobic bacteria and archaea. Unlike the class I and II RNRs, where reducing equivalents for the reaction are delivered by a redoxin (thioredoxin, glutaredoxin, or NrdH) via a pair of conserved active site cysteines, the class III RNRs examined to date use formate as the reductant. Here, we report that reaction of the Escherichia coli class III RNR with CTP (substrate) and ATP (allosteric effector) in the absence of formate leads to loss of the G• concomitant with stoichiometric formation of a new radical species and a “trapped” cytidine derivative that can break down to cytosine. Addition of formate to the new species results in recovery of 80% of the G• and reduction of the cytidine derivative, proposed to be 3′-keto-deoxycytidine, to dCTP and a small amount of cytosine. The structure of the new radical has been identified by 9.5 and 140 GHz EPR spectroscopy on isotopically labeled varieties of the protein to be a thiosulfuranyl radical [RSSR[subscript 2]]•, composed of a cysteine thiyl radical stabilized by an interaction with a methionine residue. The presence of a stable radical species on the reaction pathway rationalizes the previously reported [[superscript 3]H]-(k[subscript cat]/K[subscript M]) isotope effect of 2.3 with [[superscript 3]H]-formate, requiring formate to exchange between the active site and solution during nucleotide reduction. Analogies with the disulfide anion radical proposed to provide the reducing equivalent to the 3′-keto-deoxycytidine intermediate by the class I and II RNRs provide further evidence for the involvement of thiyl radicals in the reductive half-reaction catalyzed by all RNRs.NWO of the Netherlands (Rubicon Fellowship)Singapore. Agency for Science, Technology and ResearchNational Institutes of Health (U.S.) (Grant GM29595)National Institutes of Health (U.S.) (Grant EB-002804)National Institutes of Health (U.S.) (Grant EB-002026

Off-resonance NOVEL

Dynamic nuclear polarization (DNP) is theoretically able to enhance the signal in nuclear magnetic resonance (NMR) experiments by a factor γ[subscript e]/γ[subscript n], where γ's are the gyromagnetic ratios of an electron and a nuclear spin. However, DNP enhancements currently achieved in high-field, high-resolution biomolecular magic-angle spinning NMR are well below this limit because the continuous-wave DNP mechanisms employed in these experiments scale as ω[superscript -n over subscript 0] where n ∼ 1-2. In pulsed DNP methods, such as nuclear orientation via electron spin-locking (NOVEL), the DNP efficiency is independent of the strength of the main magnetic field. Hence, these methods represent a viable alternative approach for enhancing nuclear signals. At 0.35 T, the NOVEL scheme was demonstrated to be efficient in samples doped with stable radicals, generating [superscript 1]H NMR enhancements of ∼430. However, an impediment in the implementation of NOVEL at high fields is the requirement of sufficient microwave power to fulfill the on-resonance matching condition, ω0I = ω1S, where ω[subscript 0I] and ω[subscript 1S] are the nuclear Larmor and electron Rabi frequencies, respectively. Here, we exploit a generalized matching condition, which states that the effective Rabi frequency, ω[superscript eff over subscript 1S], matches ω[subscript 0I]. By using this generalized off-resonance matching condition, we generate [superscript 1]H NMR signal enhancement factors of 266 (∼70% of the on-resonance NOVEL enhancement) with ω[subscript 1S]/2π = 5 MHz. We investigate experimentally the conditions for optimal transfer of polarization from electrons to [superscript 1]H both for the NOVEL mechanism and the solid-effect mechanism and provide a unified theoretical description for these two historically distinct forms of DNP.National Institutes of Biomedical Imaging and Bioengineering (grant nos. EB-002804 and EB-002026

Off-resonance NOVEL

Dynamic nuclear polarization (DNP) is theoretically able to enhance the signal in nuclear magnetic resonance (NMR) experiments by a factor γe/γn, where γ’s are the gyromagnetic ratios of an electron and a nuclear spin. However, DNP enhancements currently achieved in high-field, high-resolution biomolecular magic-angle spinning NMR are well below this limit because the continuous-wave DNP mechanisms employed in these experiments scale as ω−n0 where n ~ 1–2. In pulsed DNP methods, such as nuclear orientation via electron spin-locking (NOVEL), the DNP efficiency is independent of the strength of the main magnetic field. Hence, these methods represent a viable alternative approach for enhancing nuclear signals. At 0.35 T, the NOVEL scheme was demonstrated to be efficient in samples doped with stable radicals, generating 1H NMR enhancements of ~430. However, an impediment in the implementation of NOVEL at high fields is the requirement of sufficient microwave power to fulfill the on-resonance matching condition, ω0I = ω1S, where ω0I and ω1S are the nuclear Larmor and electron Rabi frequencies, respectively. Here, we exploit a generalized matching condition, which states that the effective Rabi frequency, ωeff1S, matches ω0I. By using this generalized off-resonance matching condition, we generate 1H NMR signal enhancement factors of 266 (~70% of the on-resonance NOVEL enhancement) with ω1S/2π = 5 MHz. We investigate experimentally the conditions for optimal transfer of polarization from electrons to 1H both for the NOVEL mechanism and the solid-effect mechanism and provide a unified theoretical description for these two historically distinct forms of DNP.publishe

Simulation and design of shaped pulses beyond the piecewise-constant approximation

Response functions of resonant circuits create ringing artefacts if their input changes rapidly. When physical limits of electromagnetic spectroscopies are explored, this creates two types of problems. Firstly, simulation: the system must be propagated accurately through every response transient, this may be computationally expensive. Secondly, optimal control: circuit response must be taken into account; it may be advantageous to design pulses that are resilient to such distortions. At the root of both problems is the popular piecewise-constant approximation for control sequences in the rotating frame; in magnetic resonance it has persisted since the earliest days and has become entrenched in the commercially available hardware. In this paper, we report an implementation and benchmarks of recent Lie-group methods that can efficiently simulate and optimise smooth control sequences

Time-optimized pulsed dynamic nuclear polarization

Pulsed dynamic nuclear polarization (DNP) techniques can accomplish electron-nuclear polarization transfer efficiently with an enhancement factor that is independent of the Zeeman field. However, they often require large Rabi frequencies and, therefore, high-power microwave irradiation. Here, we propose a new low-power DNP sequence for static samples that is composed of a train of microwave pulses of length τp spaced with delays d. A particularly robust DNP condition using a period τm = τp + d set to ~1.25 times the Larmor period τLarmor is investigated which is a time-optimized pulsed DNP sequence (TOP-DNP). At 0.35 T, we obtained an enhancement of ~200 using TOP-DNP compared to ~172 with nuclear spin orientation via electron spin locking (NOVEL), a commonly used pulsed DNP sequence, while using only ~7% microwave power required for NOVEL. Experimental data and simulations at higher fields suggest a field-independent enhancement factor, as predicted by the effective Hamiltonian.publishe

Exploring the Fe(III) binding sites of human serum transferrin with EPR at 275 GHz

We report 275 GHz EPR spectra of human serum transferrin. At this high microwave frequency the zero-field splitting between the magnetic sublevels of the high-spin Fe3+Fe3+ sites can be accurately determined. We find the zero-field splitting to be a sensitive probe of the structure of the transferrin iron-binding sites. Signals arising from iron bound to the transferrin N-lobe can clearly be distinguished from signals from iron bound to the C-lobe. Moreover, our spectra show that the structure of the iron site in the N-lobe is influenced by the presence and conformation of the C-lobe. The spectra of a series of N-lobe mutants altering the second-shell interaction of Arg124 with the synergistic anion carbonate reflect conformational changes induced at the iron site.Netherlands Organization for Scientific Research (NWO) (Department of Chemical Sciences (CW)

Correction: Conformation of bis-nitroxide polarizing agents by multi-frequency EPR spectroscopy

ISSN:1463-9084ISSN:1463-907

Pulsed Dynamic Nuclear Polarization with Trityl Radicals

Continuous-wave (CW) dynamic nuclear polarization (DNP) is now established as a method of choice to enhance the sensitivity in a variety of NMR experiments. Nevertheless, there remains a need for the development of more efficient methods to transfer polarization from electrons to nuclei. Of particular interest are pulsed DNP methods because they enable a rapid and efficient polarization transfer that, in contrast with CW DNP methods, is not attenuated at high magnetic fields. Here we report nuclear spin orientation via electron spin-locking (NOVEL) experiments using the polarizing agent trityl OX063 in glycerol/water at a temperature of 80 K and a magnetic field of 0.34 T. [superscript 1]H NMR signal enhancements up to 430 are observed, and the buildup of the local polarization occurs in a few hundred nanoseconds. Thus, NOVEL can efficiently dynamically polarize [superscript 1]H atoms in a system that is of general interest to the solid-state DNP NMR community. This is a first, important step toward the general application of pulsed DNP at higher fields.National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grants EB-002026 and EB-002804)Netherlands Organization for Scientific Research (Rubicon Fellowship

Conformation of bis-nitroxide polarizing agents by multi-frequency EPR spectroscopy

The chemical structure of polarizing agents critically determines the efficiency of dynamic nuclear polarization (DNP). For cross-effect DNP, biradicals are the polarizing agents of choice and the interaction and relative orientation of the two unpaired electrons should be optimal. Both parameters are affected by the molecular structure of the biradical in the frozen glassy matrix that is typically used for DNP/MAS NMR and likely differs from the structure observed with X-ray crystallography. We have determined the conformations of six bis-nitroxide polarizing agents, including the highly efficient AMUPol, in their DNP matrix with EPR spectroscopy at 9.7 GHz, 140 GHz, and 275 GHz. The multi-frequency approach in combination with an advanced fitting routine allows us to reliably extract the interaction and relative orientation of the nitroxide moieties. We compare the structures of six bis-nitroxides to their DNP performance at 500 MHz/330 GHz.National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002804)National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002026)National Institute of General Medical Sciences (U.S.) (Award GM-095843