Probing the electronic structure and hydride occupancy in barium titanium oxyhydride through DFT-assisted solid-state NMR.

Perovskite-type oxhydrides such as BaTiO3-xHy exhibit mixed hydride ion and electron conduction and are an attractive class of materials for developing energy storage devices. However, the underlying mechanism of electric conductivity and its relation to the composition of the material remains unclear. Here we report detailed insights into the hydride local environment, the electronic structure and hydride conduction dynamics of barium titanium oxyhydride. We demonstrate that DFT-assisted solid-state NMR is an excellent tool for differentiating between the different feasible electronic structures in these solids. Our results indicate that upon reduction of BaTiO3 the introduced electrons are delocalized among all Ti atoms forming a bandstate. Furthermore, each vacated anion site is reoccupied by at most a single hydride, or else remains vacant. This single occupied bandstate structure persists at different hydrogen concentrations (y = 0.13-0.31) and a wide range of temperatures (∼100-300 K).

Separation of quadrupolar and paramagnetic shift interactions in high-resolution nuclear magnetic resonance of spinning powders.

Separation and correlation of the shift anisotropy and the first-order quadrupolar interaction of spin I = 1 nuclei under magic-angle spinning (MAS) are achieved by the phase-adjusted spinning sideband (PASS) nuclear magnetic resonance (NMR) experiment. Compared to methods for static samples, this approach has the benefit of higher sensitivity and resolution. Moreover, the PASS experiment has the advantage over previous MAS sequences in the ability to completely separate the shift anisotropy and first-order quadrupolar interactions. However, the main drawback of the pulse sequence is the lower excitation bandwidth. The sequence is comprehensively evaluated using theoretical calculations and numerical simulations and applied experimentally to the 2H NMR of a range of paramagnetic systems: deuterated nickel(II) acetate tetrahydrate, deuterated copper(II) chloride dihydrate, and two forms of deuterated oxyhydride ion conductor BaTiO3-xHy. Our results show that despite the issue with broadband excitation, the extracted shift and quadrupolar interaction tensors and the Euler angles relating the two tensors match well with the NMR parameters obtained with static NMR methods. Therefore, the new application of the PASS experiment is an excellent addition to the arsenal of NMR experiments for 2H and potentially 14N in paramagnetic solids.

Low-power synchronous helical pulse sequences for large anisotropic interactions in MAS NMR: Double-quantum excitation of 14N.

We develop a theoretical framework for a class of pulse sequences in the nuclear magnetic resonance (NMR) of rotating solids, which are applicable to nuclear spins with anisotropic interactions substantially larger than the spinning frequency, under conditions where the radiofrequency amplitude is smaller than or comparable to the spinning frequency. The treatment is based on average Hamiltonian theory and allows us to derive pulse sequences with well-defined relationships between the pulse parameters and spinning frequency for exciting specific coherences without the need for any detailed calculations. This framework is applied to the excitation of double-quantum spectra of 14N and is used both to evaluate the existing low-power pulse schemes and to predict the new ones, which we present here. It is shown that these sequences can be designed to be γ-encoded and therefore allow the acquisition of sideband-free spectra. It is also shown how these new double-quantum excitation sequences are incorporated into heteronuclear correlation NMR, such as 1H-14N dipolar double-quantum heteronuclear multiple-quantum correlation spectroscopy. The new experiments are evaluated both with numerical simulations and experiments on glycine and N-acetylvaline, which represent cases with "moderate" and "large" quadrupolar interactions, respectively. The analyzed pulse sequences perform well for the case of a "moderate" quadrupolar interaction, however poorly with a "large" quadrupolar interaction, for which future work on pulse sequence development is necessary.

High-yield Production of Amyloid-ß Peptide Enabled by a Customized Spider Silk Domain.

During storage in the silk gland, the N-terminal domain (NT) of spider silk proteins (spidroins) keeps the aggregation-prone repetitive region in solution at extreme concentrations. We observe that NTs from different spidroins have co-evolved with their respective repeat region, and now use an NT that is distantly related to previously used NTs, for efficient recombinant production of the amyloid-ß peptide (Aß) implicated in Alzheimer's disease. A designed variant of NT from Nephila clavipes flagelliform spidroin, which in nature allows production and storage of ß-hairpin repeat segments, gives exceptionally high yields of different human Aß variants as a solubility tag. This tool enables efficient production of target peptides also in minimal medium and gives up to 10 times more isotope-labeled monomeric Aß peptides per liter bacterial culture than previously reported.

Artefact-free broadband 2D NMR for separation of quadrupolar and paramagnetic shift interactions.

Two new two-dimensional, broadband, solid-state NMR experiments for separating and correlating the quadrupolar and shift interactions of spin I=1 nuclei in paramagnetic systems are proposed. The new pulse sequences incorporate the short, high-power adiabatic pulses (SHAPs) into the shifting d-echo experiment of Walder et al. [J. Chem. Phys., 142, 014201 (2015)], in two different ways, giving double and quadruple adiabatic shifting d-echo sequences. These new experiments have the advantage over previous methods of both suppressing spectral artefacts due to pulse imperfections, and exhibiting a broader excitation bandwidth. Both experiments are analysed with theoretical calculations and simulations, and are applied experimentally to the 2H NMR of deuterated CuCl2 ⋅2H2O, and two deuterated samples of the ion conductor oxyhydride BaTiO3-xHy prepared using two different methods. For the CuCl2 ⋅2H2O sample, both new methods obtain very high-quality spectra from which the parameters describing the shift and quadrupolar interaction tensors, and their relative orientation, were extracted. The two BaTiO3-xHy samples exhibited different local hydride environments with different tensor parameters. The 2H spectra of these oxyhydrides exhibit inhomogeneous broadening of the 2H shifts, and so whilst the quadrupolar interaction parameters were easily extracted, the measurement of the shift parameters was more complex. However, effective shift parameters were extracted, which combine the effects of both the paramagnetic shift tensor and the inhomogeneous broadening.

A spidroin-derived solubility tag enables controlled aggregation of a designed amyloid protein.

Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation-prone peptides and proteins at high yields. Herein, we investigate whether fusion to NT* can prevent amyloid fibril formation and enable controlled aggregation for experimental studies. As an example of an amyloidogenic protein, we chose the de novo-designed polypeptide ß17. The fusion protein NT*-ß17 was recombinantly expressed in Escherichia coli to produce high amounts of soluble and mostly monomeric protein. Structural analysis showed that ß17 is kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, ß17 adopts a ß-sheet conformation in a pH- and salt-dependent manner and assembles into amyloid-like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases.

N-Leucinyl Benzenesulfonamides as Structurally Simplified Leucyl-tRNA Synthetase Inhibitors.

N-Leucinyl benzenesulfonamides have been discovered as a novel class of potent inhibitors of E. coli leucyl-tRNA synthetase. The binding of inhibitors to the enzyme was measured by using isothermal titration calorimetry. This provided information on enthalpy and entropy contributions to binding, which, together with docking studies, were used for structure-activity relationship analysis. Enzymatic assays revealed that N-leucinyl benzenesulfonamides display remarkable selectivity for E. coli leucyl-tRNA synthetase compared to S. aureus and human orthologues. The simplest analogue of the series, N-leucinyl benzenesulfonamide (R = H), showed the highest affinity against E. coli leucyl-tRNA synthetase and also exhibited antibacterial activity against Gram-negative pathogens (the best MIC = 8 µg/mL, E. coli ATCC 25922), which renders it as a promising template for antibacterial drug discovery.

Structural studies of amyloid-ß peptides: Unlocking the mechanism of aggregation and the associated toxicity.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases worldwide. Formation of amyloid plaques consisting of amyloid-ß peptides (Aß) is one of the hallmarks of AD. Several lines of evidence have shown a correlation between the Aß aggregation and the disease development. Extensive research has been conducted with the aim to reveal the structures of the neurotoxic Aß aggregates. However, the exact structure of pathological aggregates and mechanism of the disease still remains elusive due to complexity of the occurring processes and instability of various disease-relevant Aß species. In this article we review up-to-date structural knowledge about amyloid-ß peptides, focusing on data acquired using solution and solid state NMR techniques. Furthermore, we discuss implications from these structural studies on the mechanisms of aggregation and neurotoxicity.

Lunasin is a redox sensitive intrinsically disordered peptide with two transiently populated α-helical regions.

Lunasin is a 43 amino acid peptide with anti-cancer, antioxidant, anti-inflammatory and cholesterol-lowering properties. Although the mechanism of action of lunasin has been characterized to some extent, its exact three-dimensional structure as well as the function of the N-terminal sequence remains unknown. We established a novel method for the production of recombinant lunasin that allows efficient isotope labeling for NMR studies. Initial studies showed that lunasin can exist in a reduced or oxidized state with an intramolecular disulfide bond depending on solution conditions. The structure of both forms of the peptide at pH 3.5 and 6.5 was characterized by CD spectroscopy and multidimensional NMR methods. The data indicate that lunasin belongs to the class of intrinsically disordered proteins. The analysis of secondary structure propensities indicates the presence of two helical regions and an extended (beta strand) conformation at the C-terminus. We hypothesize that the transient secondary structure elements could be stabilized upon interaction with the histones H3 and H4. The newly discovered redox properties of lunasin could explain its antioxidant and anti-inflammatory activity.