Colloidal pathways of amorphous calcium carbonate formation lead to distinct water environments and conductivity.

CaCO3 is the most abundant biomineral and a major constituent of incrustations arising from water hardness. Polycarboxylates play key roles in controlling mineralization. Herein, we present an analytical and spectroscopic study of polycarboxylate-stabilized amorphous CaCO3 (ACC) and its formation via a dense liquid precursor phase (DLP). Polycarboxylates facilitate pronounced, kinetic bicarbonate entrapment in the DLP. Since bicarbonate is destabilized in the solid state, DLP dehydration towards solid ACC necessitates the formation of locally calcium deficient sites, thereby inhibiting nucleation. Magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy of poly-aspartate-stabilized ACC reveals the presence of two distinct environments. The first contains immobile calcium and carbonate ions and structural water molecules, undergoing restricted, anisotropic motion. In the second environment, water molecules undergo slow, but isotropic motion. Indeed, conductive atomic force microscopy (C-AFM) reveals that ACC conducts electrical current, strongly suggesting that the mobile environment pervades the bulk of ACC, with dissolved hydroxide ions constituting the charge carriers. We propose that the distinct environments arise from colloidally stabilized interfaces of DLP nanodroplets, consistent with the pre-nucleation cluster (PNC) pathway.

Dynamic nuclear polarization by two-pulse phase modulation.

The coherent transfer of electron spin polarization to nuclei by means of a microwave pulse sequence is a promising new approach to enhancing the sensitivity of solid-state nuclear magnetic resonance (NMR). The development of pulse sequences for dynamic nuclear polarization (DNP) of bulk nuclei is far from complete, as is the understanding of what makes a good DNP sequence. In this context, we introduce a new sequence, termed Two-Pulse Phase Modulation (TPPM) DNP. We provide a general theoretical description for electron-proton polarization transfer by periodic DNP pulse sequences and find it in excellent agreement with numerical simulations. In experiments at 1.2 T, TPPM DNP generates a higher gain in sensitivity than existing sequences XiX (X-inverse-X) and TOP (Time-Optimized Pulsed) DNP but does so at relatively high nutation frequencies. In contrast, we find that the XiX sequence performs very well at nutation frequencies as low as 7 MHz. A combination of theoretical analysis and experimental investigation makes clear that fast electron-proton polarization transfer, due to a well-preserved dipolar coupling in the effective Hamiltonian, correlates with a short build-up time of the dynamic nuclear polarization of the bulk. Experiments further show that the performances of XiX and TOP DNP are affected differently by the concentration of the polarizing agent. These results constitute important reference points for the development of new and better DNP sequences.

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.

Anle138b interaction in α-synuclein aggregates by dynamic nuclear polarization NMR.

Small molecules that bind to oligomeric protein species such as membrane proteins and fibrils are of clinical interest for development of therapeutics and diagnostics. Definition of the binding site at atomic resolution via NMR is often challenging due to low binding stoichiometry of the small molecule. For fibrils and aggregation intermediates grown in the presence of lipids, we report atomic-resolution contacts to the small molecule at sub nm distance via solid-state NMR using dynamic nuclear polarization (DNP) and orthogonally labelled samples of the protein and the small molecule. We apply this approach to α-synuclein (αS) aggregates in complex with the small molecule anle138b, which is a clinical drug candidate for disease modifying therapy. The small central pyrazole moiety of anle138b is detected in close proximity to the protein backbone and differences in the contacts between fibrils and early intermediates are observed. For intermediate species, the 100 K condition for DNP helps to preserve the aggregation state, while for both fibrils and oligomers, the DNP enhancement is essential to obtain sufficient sensitivity.

Efficient Pulsed Dynamic Nuclear Polarization with the X-Inverse-X Sequence.

Pulsed dynamic nuclear polarization (DNP) is a promising new approach to enhancing the sensitivity of high-resolution magic-angle spinning (MAS) NMR. In pulsed DNP, the transfer of polarization from unpaired electrons to nuclei (usually 1H) is induced by a sequence of microwave pulses. Enhancement factors of the thermal 1H polarization are expected to be independent of the magnetic field, and sample heating by absorption of microwave irradiation will be strongly reduced. The development of DNP pulse sequences is still in its infancy. Of the two basic sequences in existence, NOVEL and TOP DNP, the former is, due to an extremely high power requirement, incompatible with high-resolution MAS NMR, while the latter displays a relatively slow transfer of polarization from electrons to 1H. We introduce here a new pulse sequence for DNP of solids, termed X-inverse-X (XiX) DNP. In experiments at 1.2 T, XiX DNP produces, compared to TOP DNP, a 2-fold higher gain in sensitivity. Our data suggest that a faster transfer of polarization from electrons to 1H is behind the superior performance of XiX DNP. Numerical simulations and experiments indicate that microwave pulse lengths can be chosen across a broad range, without loss of efficiency. These findings are a substantial step toward the implementation of pulsed DNP at high magnetic fields.

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

Correction for 'Conformation of bis-nitroxide polarizing agents by multi-frequency EPR spectroscopy' by Janne Soetbeer et al., Phys. Chem. Chem. Phys., 2018, 20, 25506-25517.

Analysis of the EPR spectra of transferrin: the importance of a zero-field-splitting distribution and 4th-order terms.

Multi-frequency EPR spectroscopy can provide high-level structural information on high-spin Fe3+ sites in proteins and enzymes. Unfortunately, analysis of the EPR spectra of these spin systems is hindered by the presence of broad distributions in the zero-field-splitting (ZFS) parameters, which reflect conformational heterogeneity of the iron sites. We present the analysis of EPR spectra of high-spin Fe3+ bound to human serum transferrin. We apply a method termed the grid-of-errors to extract the distributions of the individual ZFS parameters from EPR spectra recorded in the high-field limit at a microwave frequency of 275 GHz. Study of a series of transferrin variants shows that the ZFS distributions are as characteristic of the structure of a high-spin Fe3+ site as the ZFS parameters themselves. Simulations based on the extracted ZFS distributions reproduce spectra recorded at 34 GHz (Q band) and 9.7 GHz (X band), including subtle variations that were previously difficult to quantify. The X-band spectrum of transferrin shows a characteristic double peak, which has puzzled researchers for decades. We show that the double peak is uniquely related to the term B4-3O4-3(S) in the spin Hamiltonian. Our method is generally applicable in the analysis of spectra that arise from a broad distribution of parameters.

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.

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.

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 ω0-n 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, ω1Seff, 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.

Efficient cross-effect dynamic nuclear polarization without depolarization in high-resolution MAS NMR.

Dynamic nuclear polarization (DNP) has the potential to enhance the sensitivity of magic-angle spinning (MAS) NMR by many orders of magnitude and therefore to revolutionize atomic resolution structural analysis. Currently, the most widely used approach to DNP for studies of chemical, material, and biological systems involves the cross-effect (CE) mechanism, which relies on biradicals as polarizing agents. However, at high magnetic fields (≥5 T), the best biradicals used for CE MAS-DNP are still far from optimal, primarily because of the nuclear depolarization effects they induce. In the presence of bisnitroxide biradicals, magic-angle rotation results in a reverse CE that can deplete the initial proton Boltzmann polarization by more than a factor of 2. In this paper we show that these depolarization losses can be avoided by using a polarizing agent composed of a narrow-line trityl radical tethered to a broad-line TEMPO. Consequently, we show that a biocompatible trityl-nitroxide biradical, TEMTriPol-1, provides the highest MAS NMR sensitivity at ≥10 T, and its relative efficiency increases with the magnetic field strength. We use numerical simulations to explain the absence of depolarization for TEMTriPol-1 and its high efficiency, paving the way for the next generation of polarizing agents for DNP. We demonstrate the superior sensitivity enhancement using TEMTriPol-1 by recording the first solid-state 2D 13C-13C correlation spectrum at natural isotopic abundance at a magnetic field of 18.8 T.

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. (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 (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.

Efficient Dynamic Nuclear Polarization at 800 MHz/527 GHz with Trityl-Nitroxide Biradicals.

Cross-effect (CE) dynamic nuclear polarization (DNP) is a rapidly developing technique that enhances the signal intensities in magic-angle spinning (MAS) NMR spectra. We report CE DNP experiments at 211, 600, and 800 MHz using a new series of biradical polarizing agents referred to as TEMTriPols, in which a nitroxide (TEMPO) and a trityl radical are chemically tethered. The TEMTriPol molecule with the optimal performance yields a record (1) H NMR signal enhancement of 65 at 800 MHz at a concentration of 10 mM in a glycerol/water solvent matrix. The CE DNP enhancement for the TEMTriPol biradicals does not decrease as the magnetic field is increased in the manner usually observed for bis-nitroxides. Instead, the relatively strong exchange interaction between the trityl and nitroxide moieties determines the magnetic field at which the optimum enhancement is observed.

Methane to acetic acid over Cu-exchanged zeolites: mechanistic insights from a site-specific carbonylation reaction.

The selective low temperature oxidation of methane is an attractive yet challenging pathway to convert abundant natural gas into value added chemicals. Copper-exchanged ZSM-5 and mordenite (MOR) zeolites have received attention due to their ability to oxidize methane into methanol using molecular oxygen. In this work, the conversion of methane into acetic acid is demonstrated using Cu-MOR by coupling oxidation with carbonylation reactions. The carbonylation reaction, known to occur predominantly in the 8-membered ring (8MR) pockets of MOR, is used as a site-specific probe to gain insight into important mechanistic differences existing between Cu-MOR and Cu-ZSM-5 during methane oxidation. For the tandem reaction sequence, Cu-MOR generated drastically higher amounts of acetic acid when compared to Cu-ZSM-5 (22 vs 4 µmol/g). Preferential titration with sodium showed a direct correlation between the number of acid sites in the 8MR pockets in MOR and acetic acid yield, indicating that methoxy species present in the MOR side pockets undergo carbonylation. Coupled spectroscopic and reactivity measurements were used to identify the genesis of the oxidation sites and to validate the migration of methoxy species from the oxidation site to the carbonylation site. Our results indicate that the Cu(II)-O-Cu(II) sites previously associated with methane oxidation in both Cu-MOR and Cu-ZSM-5 are oxidation active but carbonylation inactive. In turn, combined UV-vis and EPR spectroscopic studies showed that a novel Cu(2+) site is formed at Cu/Al <0.2 in MOR. These sites oxidize methane and promote the migration of the product to a Brønsted acid site in the 8MR to undergo carbonylation.

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 [Formula: see text] 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.

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 [RSSR2]•, 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 [(3)H]-(k(cat)/K(M)) isotope effect of 2.3 with [(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.

Reverse micelles as a platform for dynamic nuclear polarization in solution NMR of proteins.

Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly, often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein, embedded in the reverse micelle shell, and free in the aqueous core. Significant enhancements of the water resonance ranging up to ∼-93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of solution NMR spectroscopy of proteins and other biomolecules.

High-frequency EPR study of the high-spin FeII complex Fe[(SPPh2)2N]2.

We report continuous-wave electron-paramagnetic-resonance (EPR) spectra of the high-spin Fe(II) complex Fe[(SPPh(2))(2)N](2) at 275.7 GHz, 94.1 GHz and 9.5 GHz. Combined analysis of these EPR spectra shows that the complex occurs in multiple conformations. For two main conformations the spin-Hamiltonian parameters, which reflect the electronic structure of the complex, are accurately determined: (1) D=9.17 cm(-1) (275 GHz), E/D=0.021 and (2) D=8.87 cm(-1) (266 GHz), E/D=0.052. The EPR spectra obtained at 275.7 GHz on single crystals of the complex are essential for the analysis and in addition they reveal that the two main conformations occur at two magnetically distinguishable sites.

Multifrequency EPR study of Fe3+ and Co2+ in the active site of desulforedoxin.

The understanding of the electronic structure of S > 1/2 transition-metal sites that show a large zero-field splitting (ZFS) of the magnetic sublevels benefits greatly from study by electron-paramagnetic-resonance (EPR) spectroscopy at frequencies above the standard 9.5 GHz. However, high-frequency EPR spectroscopy is technically challenging and still developing. Particularly the sensitivity of high-frequency EPR spectrometers is often too low to apply the technique in the study of transition-metal sites in proteins and enzymes. Here we report a multifrequency EPR study (at 9.5, 94.9, and 275.7 GHz) of the active site of the protein desulforedoxin, both in its natural Fe(3+) form and substituted with Co(2+). The 275.7 GHz EPR spectra made it possible to determine the ZFS parameters of the Fe(3+) site with high precision. No 275.7 GHz spectrum could be observed of the Co(2+) site, but based on 9.5 GHz spectra, its ZFS parameters could be estimated. We find that the typical variation in the geometry of the active site of a protein or enzyme, referred to as conformational strain, does not only make the detection of EPR spectra challenging, but also their analysis. Comparison of the EPR results on the active site of desulforedoxin to those of the closely related active site of rubredoxin illustrates the necessity of explicit quantum-chemical calculations in order to interrelate the electronic and geometric structure of biological transition-metal sites.

Configuration of spheroidene in the photosynthetic reaction center of Rhodobacter sphaeroides : a comparison of wild-type and reconstituted R26.

We compare the resonance Raman spectra acquired at two excitation wavelengths, 496.5 and 514.5 nm, of spheroidene in the wild-type reaction center of Rhodobacter sphaeroides and reconstituted into the reaction center of the Rhodobacter sphaeroides mutant R26. Our earlier work showed that the reconstituted R26 reaction center holds spheroidene in two configurations: 15,15'-cis and another configuration. Here we show that in the wild-type reaction center only 15,15'-cis spheroidene is present. In the resonance Raman spectra of the reconstituted R26 reaction centers, a transition is identified that arises exclusively from the second configuration. According to density-functional-theory calculations, this transition is specific for the 13,14-cis configuration.