NMR Signatures and Electronic Structure of Ti Sites in Titanosilicalite-1 from Solid-State 47/49Ti NMR Spectroscopy.

Although titanosilicalite-1 (TS-1) is among the most successful oxidation catalysts used in industry, its active site structure is still debated. Recent efforts have mostly focused on understanding the role of defect sites and extraframework Ti. Here, we report the 47/49Ti signature of TS-1 and molecular analogues [Ti(OTBOS)4] and [Ti(OTBOS)3(OiPr)] using novel MAS CryoProbe to enhance the sensitivity. While the dehydrated TS-1 displays chemical shifts similar to those of molecular homologues, confirming the tetrahedral environment of Ti consistent with X-ray absorption spectroscopy, it is associated with a distribution of larger quadrupolar coupling constants, indicating an asymmetric environment. Detailed computational studies on cluster models highlights the high sensitivity of the NMR signatures (chemical shift and quadrupolar coupling constant) to small local structural changes. These calculations show that, while it will be difficult to distinguish mono- vs dinuclear sites, the sensitivity of the 47/49Ti NMR signature should enable distinguishing the Ti location among specific T site positions.

High strength, epoxy cross-linked high sulfur content polymers from one-step reactive compatibilization inverse vulcanization.

Inverse vulcanization provides a simple, solvent-free method for the preparation of high sulfur content polymers using elemental sulfur, a byproduct of refining processes, as feedstock. Despite the successful demonstration of sulfur polymers from inverse vulcanization in optical, electrochemical, and self-healing applications, the mechanical properties of these materials have remained limited. We herein report a one-step inverse vulcanization using allyl glycidyl ether, a heterobifunctional comonomer. The copolymerization, which proceeds via reactive compatibilization, gives an epoxy cross-linked sulfur polymer in a single step, as demonstrated through isothermal kinetic experiments and solid-state 13C NMR spectroscopy. The resulting high sulfur content (≥50 wt%) polymers exhibited tensile strength at break in the range of 10-60 MPa (70-50 wt% sulfur), which represents an unprecedentedly high strength for high sulfur content polymers from vulcanization. The resulting high sulfur content copolymer also exhibited extraordinary shape memory behavior along with shape reprogrammability attributed to facile polysulfide bond rearrangement.

Determining nanoform similarity via assessment of surface reactivity by abiotic and in vitro assays.

Grouping of substances is a method used to streamline hazard and risk assessment. Assessment of similarity provides the scientific evidence needed for formation of groups. This work reports on justification of grouping of nanoforms (NFs) via similarity of their surface reactivity. Four reactivity assays were used for concentration dependent detection of reactive oxygen species (ROS) generated by NFs: abiotic assays FRAS, EPR and DCFH2-DA, as well as the in vitro assay of NRF2/ARE responsive luciferase reporter activation in the HEK293 cell line. Representative materials (CuO, Mn2O3, BaSO4, CeO2 and ZnO) and three case studies of each several NFs of iron oxides, Diketopyrrolopyrroles (DPP)-based organic pigments and silicas were assessed. A novel similarity assessment algorithm was applied to quantify similarities between pairs of NFs, in a four-step workflow on concentration-response curves, individual concentration and response ranges, and finally the representative materials. We found this algorithm to be applicable to all abiotic and in vitro assays that were tested. Justification of grouping must include the increased potency of smaller particles via the scaling of effects with specific surface, and hence quantitative similarity analysis was performed on concentration-response in mass-metrics. CuO and BaSO4 were the most and least reactive representative materials respectively, and all assays found BaSO4/CuO not similar, as confirmed by their different NOAECs of in vivo studies. However, similarity outcomes from different reactivity assays were not always in agreement, highlighting the need to generate data by one assay for the representative materials and the candidate group of NFs. Despite low similarity scores in vitro some pairs of case study NFs can be accepted as sufficiently similar because the in vivo NOAECs are similar, highlighting the conservative assessment by the abiotic assays.

Adoption and diffusion of marine litter clean-up technologies across European seas: Legal, institutional and financial drivers and barriers.

This study reviews existing legal, institutional and policy tools and frameworks, relevant to the introduction and adoption of new marine litter clean-up technologies in two regional European seas, the Mediterranean and the Baltic. A combination of desk studies in six countries bordering the Baltic (Estonia, Germany, Sweden) and the Mediterranean (Greece, Italy, Tunisia), and interviews with experts and stakeholders, is used to identify key drivers and barriers to the adoption and diffusion of marine litter technologies. The main conclusion of the study is that the most influential pieces of legislation relevant to marine litter management are top-down EU policies, often forming the basis of regional and national plans. Moreover, the study finds that several drivers of marine litter technologies may at the same time be critical barriers. These factors include public awareness, consumer behaviour, enforcement of legislation, and the rise of SMEs engaged in recycling and eco-labelling of marine litter.

Probing molecular motion by double-quantum (13C,13C) solid-state NMR spectroscopy: application to ubiquitin.

We demonstrate the use of two-dimensional ((13)C,(13)C) double-quantum spectroscopy to detect molecular dynamics by solid-state NMR. Data collected on tyrosine-ethylester (TEE) are in line with previously determined ((1)H,(13)C) order parameters. Application of these experiments to microcrystalline ubiquitin reveals the presence of dynamics on millisecond or faster time scales and differences in local mobility depending on microcrystal preparation. In addition, solid-state NMR-based structure calculation indicates conformational variability of loop regions between different solid-phase ubiquitin preparations. Our data relate preparation-dependent changes observed in NMR spectral parameters such as chemical shifts and through-space correlations to differences in ubiquitin dynamics and conformation and suggest a prominent role of molecular mobility in microcrystalline ubiquitin.

Cross-examining Polyurethane Nanodomain Formation and Internal Structure.

Structural and morphological interplay between hard and soft phases determine the bulk properties of thermoplastic polyurethanes. Commonly employed techniques rely on different physical or chemical phenomena for characterizing the organization of domains, but detailed structural information can be difficult to derive. Here, total scattering pair distribution function (PDF) analysis is used to determine atomic-scale insights into the connectivity and molecular ordering and compared to the domain size and morphological characteristics measured by AFM, TEM, SAXS, WAXS, and solid-state NMR 1H-1H spin-diffusion. In particular, density distribution functions are highlighted as a means to bridging the gap from the domain morphology to intradomain structural ordering. High real-space resolution PDFs are shown to provide a sensitive fingerprint for indexing aromatic, aliphatic, and polymerization-induced bonding characteristics, as well as the hard phase structure, and indicate that hard phases coexist in both ordered and disordered states.

Structural rearrangements of membrane proteins probed by water-edited solid-state NMR spectroscopy.

We show that water-edited solid-state NMR spectroscopy allows for probing global protein conformation and residue-specific solvent accessibility in a lipid bilayer environment. The transfer dynamics can be well described by a general time constant, irrespective of protein topology and lipid environment. This approach was used to follow structural changes in response to protein function in the chimeric potassium channel KcsA-Kv1.3. Data obtained as a function of pH link earlier biochemical data to changes in protein structure in a functional bilayer setting.

Comparative analysis of NMR chemical shift predictions for proteins in the solid phase.

A comparative analysis of nuclear chemical shift predictions of proteins in the solid state by rapid algorithms trained on and verified with solution-state NMR assignments is presented. The precision of predictions by four dedicated computer programs (SHIFTS, PROSHIFTS, SHIFTX and SPARTA) was found to be close to values obtained for proteins in solution. Correlation coefficients depend on the NMR nucleus (N, C', C(alpha) and C(beta)) and on secondary structure (beta-strand, random coil and alpha-helix), but also on the molecular environment (membrane-integral or not). The findings establish a quantitative basis for using chemical shift prediction programs for solid-state NMR applications. On the other hand, prediction inaccuracies identified for certain resonance kind, residue type, and molecular environment point to possible areas of methodological improvement.

Characterization of Alzheimer's-like paired helical filaments from the core domain of tau protein using solid-state NMR spectroscopy.

The polymerization of the microtubule-associated protein tau into paired helical filaments (PHFs) represents one of the hallmarks of Alzheimer's disease. We employed solid-state nuclear magnetic resonance (NMR) to investigate the structure and dynamics of PHFs formed in vitro by the three-repeat-domain (K19) of protein tau, representing the core of Alzheimer PHFs. While N and C termini of tau monomers in PHFs are highly dynamic and solvent-exposed, the rigid segment consists of three major beta-strands. Combination of through-bond and through-space ssNMR transfer methods with water-edited ((15)N, (13)C) and ((13)C, (13)C) correlation experiments suggests the existence of a fibril core that is largely built by repeat unit R3, flanked by surface-exposed units R1 and R4. Solid-state NMR, circular dichroism, and the fibrillization behavior of a K19 mutant furthermore indicate that electrostatic interactions play a central role in stabilizing the K19 PHFs.

Ion distribution in copper exchanged zeolites by using Si-29 spin lattice relaxation analysis.

Transition metal-containing zeolites, particularly those with smaller pore size, have found extensive application in the selective catalytic reduction (SCR) of environmental pollutants containing nitrogen oxides. We report these zeolites have dramatically faster silicon-29 (Si-29) spin lattice relaxation times (T1) compared to their sodium-containing counterparts. Paramagnetic doping allows one to acquire Si-29 MAS spectra in the order of tens of seconds without significantly affecting the spectral resolution. Moreover, relaxation times depend on the method of preparation and the next-nearest neighbor silicon Qn(mAl) sites, where n=4 and m=0-4, respectively. A clear trend is noted between the effectiveness of Cu exchange and the Si-29 NMR relaxation times. It is anticipated that the availability of this tool, and the enhanced understanding of the nature of the active sites, will provide the means for designing improved SCR catalysts.

Solubilization of active ingredients of different polarity in Pluronic® micellar solutions - Correlations between solubilizate polarity and solubilization site.

The solubilization of two pharmaceutically active ingredients (AI) with significantly different water solubility, namely carbamazepine and fenofibrate (solubility of 150ppm and 10ppm, respectively), has been investigated using a series of Pluronics® (Poloxamers) containing different ethylene oxide and propylene oxide (EO/PO) units in the molecule. The results show largely enhanced solubilization of fenofibrate by Pluronic® micelles that increases with the PPO chain length provided the temperature is above the critical micelle temperature (cmt). In contrast the more water-soluble carbamazepine only shows a moderate increase in solubilization upon addition of Pluronics®. Small angle neutron scattering (SANS) and pulsed field gradient (PFG) NMR experiments show that the solubilization of fenofibrate occurs in the core of the micelles, whereas carbamazepine shows no direct association with the micelles. These clearly different solubilization mechanisms for the two AIs were confirmed by Nuclear Overhauser Enhancement Spectroscopy (NOESY) experiments, which show that fenofibrate interacts only with the PPO core of the micelle, whereas carbamazepine interacts with both PPO and PEO similarly. Accordingly, the large enhancement of the solubilization of fenofibrate is related to the fact that it is solubilized within the PPO core of the Pluronic® micelles, while the much more moderate increase of carbamazepine solubility is attributed to the change of solvent quality due to the presence of the amphiphilic copolymer and the interaction with the EO and PO units in solution.

3D NMR spectroscopy for resonance assignment and structure elucidation of proteins under MAS: novel pulse schemes and sensitivity considerations.

Two types of 3D MAS NMR experiments are introduced, which combine standard (NC,CC) transfer schemes with (1H,1H) mixing to simultaneously detect connectivities and structural constraints of uniformly 15N,13C-labeled proteins with high spectral resolution. The homonuclear CCHHC and CCC experiments are recorded with one double-quantum evolution dimension in order to avoid a cubic diagonal in the spectrum. Depending on the second transfer step, spin systems or proton-proton contacts can be determined with reduced spectral overlap. The heteronuclear NHHCC experiment encodes NH-HC proton-proton interactions, which are indicative for the backbone conformation of the protein. The third dimension facilitates the identification of the amino acid spin system. Experimental results on U-[15N,13C]valine and U-[15N,13C]ubiquitin demonstrate their usefulness for resonance assignments and for the determination of structural constraints. Furthermore, we give a detailed analysis of alternative multidimensional sampling schemes and their effect on sensitivity and resolution.

Complex formation and light activation in membrane-embedded sensory rhodopsin II as seen by solid-state NMR spectroscopy.

Microbial rhodopsins execute diverse biological functions in the cellular membrane. A mechanistic understanding of their functional profile is, however, still limited. We used solid-state NMR (ssNMR) spectroscopy to study structure and dynamics of a 2 x 400 amino acid sensory rhodopsin/transducer (SRII/HtrII) complex from Natronomonas pharaonis in a natural membrane environment. We found a receptor-transducer binding interface in the ground state that significantly extends beyond the available X-ray structure. This binding domain involves the EF loop of the receptor and stabilizes the functionally relevant, directly adjacent HAMP domain of the transducer. Using 2D ssNMR difference spectroscopy, we identified protein residues that may act as a functional module around the retinal binding site during the early events of protein activation. These latter protein segments, the inherent plasticity of the HAMP domain, and the observation of an extended SRII/HtrII membrane-embedded interface may be crucial components for optimal signal relay efficiency across the cell membrane.

Protein refolding is required for assembly of the type three secretion needle.

Pathogenic Gram-negative bacteria use a type three secretion system (TTSS) to deliver virulence factors into host cells. Although the order in which proteins incorporate into the growing TTSS is well described, the underlying assembly mechanisms are still unclear. Here we show that the TTSS needle protomer refolds spontaneously to extend the needle from the distal end. We developed a functional mutant of the needle protomer from Shigella flexneri and Salmonella typhimurium to study its assembly in vitro. We show that the protomer partially refolds from alpha-helix into beta-strand conformation to form the TTSS needle. Reconstitution experiments show that needle growth does not require ATP. Thus, like the structurally related flagellar systems, the needle elongates by subunit polymerization at the distal end but requires protomer refolding. Our studies provide a starting point to understand the molecular assembly mechanisms and the structure of the TTSS at atomic level.

Structural characterization of Ca(2+)-ATPase-bound phospholamban in lipid bilayers by solid-state nuclear magnetic resonance (NMR) spectroscopy.

Phospholamban (PLN) regulates cardiac contractility by modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. While PLN and SERCA1a, an isoform from skeletal muscle, have been structurally characterized in great detail, direct information about the conformation of PLN in complex with SERCA has been limited. We used solid-state NMR (ssNMR) spectroscopy to deduce structural properties of both the A 36F 41A 46 mutant (AFA-PLN) and wild-type PLN (WT-PLN) when bound to SERCA1a after reconstitution in a functional lipid bilayer environment. Chemical-shift assignments in all domains of AFA-PLN provide direct evidence for the presence of two terminal alpha helices connected by a linker region of reduced structural order that differs from previous findings on free PLN. ssNMR experiments on WT-PLN show no significant difference in binding compared to AFA-PLN and do not support the coexistence of a significantly populated dynamic state of PLN after formation of the PLN/SERCA complex. A combination of our spectroscopic data with biophysical and biochemical data using flexible protein-protein docking simulations provides a structural basis for understanding the interaction between PLN and SERCA1a.

Studying molecular 3D structure and dynamics by high-resolution solid-state NMR: Application to L-tyrosine-ethylester.

A unified approach to the study of 3D conformation and molecular dynamics using magic-angle-spinning solid-state NMR is demonstrated on a uniformly 13C-labeled sample of L-tyrosine-ethylester.

High-resolution solid-state NMR studies on uniformly [13C,15N]-labeled ubiquitin.

Understanding of the effects of intermolecular interactions, molecular dynamics, and sample preparation on high-resolution magic-angle spinning NMR data is currently limited. Using the example of a uniformly [13C,15N]-labeled sample of ubiquitin, we discuss solid-state NMR methods tailored to the construction of 3D molecular structure and study the influence of solid-phase protein preparation on solid-state NMR spectra. A comparative analysis of 13C', 13Calpha, and 13Cbeta resonance frequencies suggests that 13C chemical-shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. Our results can be refined by additional solid-state NMR techniques and serve as a reference for ongoing efforts to characterize the structure and dynamics of (membrane) proteins, protein complexes, and other biomolecules by high-resolution solid-state NMR.

Determination of membrane protein structure and dynamics by magic-angle-spinning solid-state NMR spectroscopy.

It is shown that molecular structure and dynamics of a uniformly labeled membrane protein can be studied under magic-angle-spinning conditions. For this purpose, dipolar recoupling experiments are combined with novel through-bond correlation schemes that probe mobile protein segments. These NMR schemes are demonstrated on a uniformly [13C,15N] variant of the 52-residue polypeptide phospholamban. When reconstituted in lipid bilayers, the NMR data are consistent with an alpha-helical trans-membrane segment and a cytoplasmic domain that exhibits a high degree of structural disorder.