1H 15N Research Articles

Optimization of 15N–13C double-resonance NMR experiments under low temperature magic angle spinning dynamic nuclear polarization conditions

Dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS) solid-state NMR carried out at 25 K enables rapid acquisition of multi-dimensional 13C–15N correlation spectra for protein structure studies and resonance assignment. Under commonly used DNP conditions, solvent deuteration reduces 1H–15N cross polarization (CP) efficiencies, necessitates more careful optimization, and requires longer high-power 15N radio-frequency pulses. The sensitivity of 2D heteronuclear correlation experiments is potentially impaired. Here we show that 2D 15N-13C experiments based on 13C-15N transferred echo double resonance (TEDOR) methods outperform 2D experiments based on CP transfers in a fully deuterated solvent, and are competitive with CP-based experiments when the solvent is only partially deuterated. Additionally, we show that optimization of TEDOR-based 2D experiments is simpler than optimization of CP-based experiments under 25 K MAS conditions.

Crosslinking reactions of a model aminated lignin compound as a platform for building thermosetting polymers for lignin-based bio adhesives

As millions of tons of kraft lignin are being wasted, a potential application is to use its crosslinking reactions to build thermosetting bio adhesives. However, the crosslinking reactions between lignin molecules are not fully understood. The present study aims to elucidate the crosslinking reactions of the model lignin compound guaiacylglycerol-β-guaiacyl ether (GGE) via one-step hydroxymethylation/ amination with formaldehyde and diethylenetriamine (DETAM), or one-step glyoxylation/ amination with glyoxal and DETAM via liquid NMR techniques such as 1H NMR, 13C NMR, 2D 1H–13C, and 1H–15N HSQC NMR. Specifically, the 2D 1H–13C HSQC NMR spectra confirm the presence of –CH2–NH– with a chemical shift of 1H 2.6–3.6/13C 40–60 ppm, and the formation of methylene linkages via the crosslinking reaction. Also, the 2D 1H–15N HSQC NMR spectra clearly detect the formation of amide and imine bonds at 1H 7.8/15N 110 and 1H 8.07/15N 121.5 ppm from the crosslinked GGE.

Quantification of Hydrogen and Transition Metals in the Cathode Catalyst Layer of Anion Exchange Membrane Electrolyzer By Ion Beam Techniques

Anion Exchange Membrane Water Electrolyzer (AEMWE) has increased interest in H2 production at low cost and high scalability. The efficiency is strongly dependent on the membrane electrode assembly (MEA) with appropriate ionomer and catalyst layers. Herein, we report AEMWE with MoNi4-MoO2 cathode catalyst layer in ionomer-free Catalyst Coated Substrate (CCS) configuration. The optimized MEA for AEMWE demonstrated the superior performance of 225 mA/cm2 at 2V with more than 48 hours of durability. The X-ray diffraction confirmed the NiMoO4 phase transformation to MoNi4-MoO2 and SEM-EDS was used to study the morphology and elemental composition of the deposited catalyst. X-ray photoelectron Spectroscopy demonstrated the dominant metallic MoO2 formed after H2/Ar annealing. Rutherford Backscattering Spectroscopy (RBS) was also employed to measure the depth composition profile of the catalyst layer exhibiting the reduction of Ni concentration in the bulk after annealing. Further, we introduce hydrogen depth profiling by Nuclear Reaction Analysis (NRA) via the resonant 1H(15N,αγ)12C reaction as a versatile method for the highly depth-resolved visualization of hydrogen(H) in the near-surface region, including transitions of hydrogen between the surface and the bulk, and between shallow interfaces of the nanostructured cathode catalyst layer. The diffusion of the H atoms was observed with a 40% atomic percent of Hydrogen after H2/Ar annealing of the catalyst for 180 minutes (about 3 hours) and decreased to 10% atomic percent of hydrogen after 24 hours of electrolyzer operation. This technique also enabled the measurement of the zero-point vibrational energy of hydrogen atoms absorbed on the surfaces of the catalyst layer. Overall, this work better explains the cathode catalyst degradation effects and hydrogen diffusion in the cathode catalyst layer responsible for voltage and mass transfer losses in AEM electrolyzers.

From mother's milk to structural insights: 1H–15N NMR analysis of Lewis X antigen-bearing oligosaccharides isolated from human milk

Human milk oligosaccharides (HMOs) are complex unconjugated glycans abundant in breast milk, with vital roles in infant nutrition and health. The structural elucidation of HMOs remains challenging due to their diverse compositions and isomeric complexities. This study presents a novel approach employing 1H–15N NMR correlations to pinpoint and characterize HMOs, LNH, LNnH, and their fucosylated derivatives including those of bearing the Lewis X motif. Isolated from human milk, these hexa-, hepta-, and octasaccharides were extensively analysed by NMR. This enabled their first complete 1H, 13C and 15N resonance assignments. Using 1H–15N HSQC experiments, the distinctive 1H–15N correlations of GlcNAc units within the HMOs provided structural fingerprints, allowing for unequivocal differentiation of isomeric structures and fucosylation patterns. Results demonstrate the potential of 1H–15N NMR spectroscopy in decoding complex HMO structures, offering new perspectives on their detailed structural characterization and contributing to a deeper understanding of their potential benefits.

1Hβ chemical shift-based phase modulated NMR methods for fast identification of amino acid types in proteins

Here we report two phase modulated NMR experiments: PM-2D HN(CACBHB) and PM-2D HN(HB), that use 1Hβ chemical shifts to rapidly identify amino acid type in proteins. The magnetization on the 1Hβ spins during the experiments is allowed to evolve for a fixed evolution period that results in phase modulation (positive or negative) of the cross peaks corresponding to various amino acid residues on their 2D HN projections, resembling a typical 2D [1H–15N]-HSQC spectrum. All amino acids except glycine can be categorized into three discernible groups based on their 1Hβ chemical shifts, resulting in unique phase patterns at different fixed evolution periods for 1Hβ, thus facilitating their identification. Remarkably, the PM-2D HN(HB) stands out among all amino acid type identification NMR techniques for its applicability with cost-effective and most routinely employed 15N-labeled protein samples for NMR studies. Furthermore, when combined effectively with the 13Cβ chemical shift-based phase modulated NMR method (PM-2D HN(CACB)), these methods resolved the identification of large groups of amino acids into relatively smaller groups. Moreover, these techniques can accelerate the sequence-specific sequential resonance assignment (SSRA) process and would help in fast tracking of assigned NMR signals exhibiting chemical shift perturbation on the 2D [1H–15N]-HSQC spectrum of proteins during various experiments (e.g., temperature change, pH change, and protein or ligand or cofactor binding) as well as in site-directed mutagenesis.

Hydrogen absorption in an epitaxial thin film of high-entropy perovskite oxide

We synthesized an epitaxial film of high-entropy perovskite oxide (HEPO) consisting of three elements (Ca, Sr, Ba) in the A site and 12 elements (Si, Ti, Cr, Mn, Fe, Co, Ni, Ge, Zr, Sn, Ce, Hf) in the B site of ABO3, and investigated hydrogen absorption properties in the HEPO film. The hydrogen depth profile was measured by nuclear reaction analysis via the 1H(15N,αγ)12C reaction, showing the hydrogen absorption in the HEPO film with a maximum atomic concentration of 0.3 in the ABO3 unit. The diffusion coefficient of H in the film was analyzed from the H depth profile, and the activation barrier for diffusion was estimated to be (0.54±0.13) eV. An absorption band was observed at 3290 cm−1 in the infrared absorption spectrum, which corresponds to the O–H stretching vibration. Simultaneous measurements of nuclear reaction and resistance revealed that the HEPO film remained electrically insulating regardless of the hydrogen concentration. We demonstrated that hydrogen is incorporated in HEPO forming OH species while keeping HEPO electrically insulating.

Structural insights into the disulfide isomerase and chaperone activity of TrbB of the F plasmid type IV secretion system

Bacteria have evolved elaborate mechanisms to thrive in stressful environments. F-like plasmids in gram-negative bacteria encode for a multi-protein Type IV Secretion System (T4SSF) that is functional for bacterial proliferation and adaptation through the process of conjugation. The periplasmic protein TrbB is believed to have a stabilizing chaperone role in the T4SSF assembly, with TrbB exhibiting disulfide isomerase (DI) activity. In the current report, we demonstrate that the deletion of the disordered N-terminus of TrbBWT, resulting in a truncation construct TrbB37-161, does not affect its catalytic in vitro activity compared to the wild-type protein (p = 0.76). Residues W37–K161, which include the active thioredoxin motif, are sufficient for DI activity. The N-terminus of TrbBWT is disordered as indicated by a structural model of GST-TrbBWT based on ColabFold-AlphaFold2 and Small Angle X-Ray Scattering data and 1H–15N Heteronuclear Single Quantum Correlation (HSQC) spectroscopy of the untagged protein. This disordered region likely contributes to the protein's dynamicity; removal of this region results in a more stable protein based on 1H–15N HSQC and Circular Dichroism Spectroscopies. Lastly, size exclusion chromatography analysis of TrbBWT in the presence of TraW, a T4SSF assembly protein predicted to interact with TrbBWT, does not support the inference of a stable complex forming in vitro. This work advances our understanding of TrbB's structure and function, explores the role of structural disorder in protein dynamics in the context of a T4SSF accessory protein, and highlights the importance of redox-assisted protein folding in the T4SSF.

Green synthesis of novel pyrano[2,3-c]pyrazole-5-carbonitrile analogues by using Fe5(PW10V2O40)3 nanocatalyst through a one-pot Knoevenagel condensation and Michael addition mechanism

A swift and green one-pot method was improved for the preparation of new pyranopyrazole analogues using iron-doped heteropoly acid [(Fe5(PW10V2O40)3] (FePWV) as an efficient catalyst at room temperature and ethanol as the solvent. The one-pot, multi-component reaction combining substituted aldehyde, malononitrile,ethyl acetoacetate, and hydrazine hydrate produces remarkable yields (varying from 88 % to 98 %) for this highly selective technique. Using P-XRD, TGA, BET, FT-IR, TEM, and SEM studies, the synthesized FePWV catalyst was fully characterized. Several spectroscopic methods, including 1H NMR, 15N NMR, 13C NMR, and HRMS, were used to confirm the target molecules' structural information.This environmentally benign method also has other benefits, such as operational simplicity, a stable catalyst with great reusability (up to 7 times), quick reaction times (<20 min), and the absence of chromatographic separations. The proposed method is sustainable and affordable as a result of these properties taken together.

Synthesis, spectroscopic and structural characterizations and in vitro antimycobacterial activity of silver(I) complexes with sulfapyridine (SPY) and sulfamerazine (SM)

Sulfonamides (sulfas) are a specific class of organic compounds that possesses antibacterial, anti-inflammatory, antiviral and antitumor activities. This class of molecules is also used in the synthesis of silver(I) complexes, which usually present significant biological activity as antimicrobial agents. In this context, the present work describes the synthesis, structural characterization and antibacterial assays of two silver(I) complexes with the sulfa drugs sulfapyridine (SPY) and sulfamerazine (SM). Chemical analyses and high-resolution mass spectrometric studies led to 1:1 metal/ligand compositions, suggesting the minimal formulas C11H10N3O2SAg·2H2O for AgSPY and C11H11N4O2SAg·2H2O for AgSM. Spectroscopic techniques such as 1H, 13C and {1H–15N} nuclear magnetic resonance (NMR), and also infrared measurements (IR) suggest the coordination of the ligands to silver(I) ions by the nitrogen atom of the SO2N moiety for both complexes. Crystal structures of AgSPY and AgSM were solved and refined by X-ray powder diffraction studies, where dimeric structures with Ag(I) ions bridging between two ligand molecules by the usual eight membered ring [AgNCN]2 and strong argentophilic interactions were observed. Minimum Inhibitory Concentration (MIC90) assays showed the promising activities of the free sulfonamides and the respective complexes against Mycobacterium tuberculosis H37Rv. MIC90 values, in µmol L–1, were 2.47, 3.60, 1.33 and 1.61 for SPY, AgSPY, SM and AgSM, respectively. The obtained MIC90 values were lower than those found for silver sulfadiazine and silver nitrate in the same experimental conditions.

Hydrogen diffusion in cerium oxide thin films fabricated by pulsed laser deposition

Cerium oxide (CeO2) is well known to be reducible by hydrogen (H2), yet the diffusion and solution properties of hydrogen in ceria at elevated temperatures have remained challenging to evaluate. We therefore fabricated nanometer-thin (∼100 nm) cerium oxide films on Si(111) substrates by pulsed laser deposition (PLD) and quantitatively investigated the H depth distributions therein by means of resonant 1H(15N,αγ)12C nuclear reaction analysis (NRA) before and after annealing in H2 gas at 773–973 K. X-ray diffraction (XRD), x-ray photoelectron spectroscopy (XPS) and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS) reveal that the as-deposited films exhibit single phase CeO2 structure and partially reduced stoichiometry (CeO1.69). H2 annealing does not largely change the H content of the as-deposited films; in all conditions several atomic percent of hydroxyl (OH) are found to exist in a thin (∼4 nm) surface layer, whereas stably bound hydrogen in the bulk of the films is almost uniformly distributed and of much smaller quantity (∼0.2 at.%) than the oxygen vacancy concentration in the partially reduced ceria. Its low concentration and high thermal stability identify this bulk H species as likely being strongly bound to defects in the polycrystalline films rather than as a hydride species that interacts weakly with O-vacancies. The H diffusion coefficient and activation energy in the ceria films are determined as > 10−18 m2 s−1 at 773–973 K and <1.69 eV, respectively. The observed diffusion activation energy is somewhat larger than theoretical predictions for thermal diffusion of H in stoichiometric bulk CeO2, suggesting that defects and oxygen vacancies in the PLD-fabricated ceria thin films possibly have an impact on the H mobility.

Biochemical and biophysical characterization of the RAS family small GTPase protein DiRAS3

DiRAS3, also called ARHI, is a RAS (sub)family small GTPase protein that shares 50–60% sequence identity with H-, K-, and N-RAS, with substitutions in key conserved G-box motifs and a unique 34 amino acid extension at its N-terminus. Unlike the RAS proto-oncogenes, DiRAS3 exhibits tumor suppressor properties. DiRAS3 function has been studied through genetics and cell biology, but there has been a lack of understanding of the biochemical and biophysical properties of the protein, likely due to its instability and poor solubility. To overcome this solubility issue, we engineered a DiRAS3 variant (C75S/C80S), which significantly improved soluble protein expression in E. coli. Recombinant DiRAS3 was purified by Ni-NTA and size exclusion chromatography (SEC). Concentration dependence of the SEC chromatogram indicated that DiRAS3 exists in monomer-dimer equilibrium. We then produced truncations of the N-terminal (ΔN) and both (ΔNC) extensions to the GTPase domain. Unlike full-length DiRAS3, the SEC profiles showed that ΔNC is monomeric while ΔN was monomeric with aggregation, suggesting that the N and/or C-terminal tail(s) contribute to dimerization and aggregation. The 1H–15N HSQC NMR spectrum of ΔNC construct displayed well-dispersed peaks similar to spectra of other GTPase domains, which enabled us to demonstrate that DiRAS3 has a GTPase domain that can bind GDP and GTP. Taken together, we conclude that, despite the substitutions in the G-box motifs, DiRAS3 can switch between nucleotide-bound states and that the N- and C-terminal extensions interact transiently with the GTPase domain in intra- and inter-molecular fashions, mediating weak multimerization of this unique small GTPase.

Site-specific protein backbone deuterium 2Hα quadrupolar patterns by proton-detected quadruple-resonance 3D 2HαcαNH MAS NMR spectroscopy

A novel deuterium-excited and proton-detected quadruple-resonance three-dimensional (3D) 2HαcαNH MAS nuclear magnetic resonance (NMR) method is presented to obtain site-specific 2Hα deuterium quadrupolar couplings from protein backbone, as an extension to the 2D version of the experiment reported earlier. Proton-detection results in high sensitivity compared to the heteronuclei detection methods. Utilizing four independent radiofrequency (RF) channels (quadruple-resonance), we managed to excite the 2Hα, then transfer deuterium polarization to its attached Cα, followed by polarization transfers to the neighboring backbone nitrogen and then to the amide proton for detection. This experiment results in an easy to interpret HSQC-like 2D 1H–15N fingerprint NMR spectrum, which contains site-specific deuterium quadrupolar patterns in the indirect third dimension. Provided that four-channel NMR probe technology is available, the setup of the 2HαcαNH experiment is relatively straightforward, by using low power deuterium excitation and polarization transfer schemes we have been developing. To our knowledge, this is the first demonstration of a quadruple-resonance MAS NMR experiment to link 2Hα quadrupolar couplings to proton-detection, extending our previous triple-resonance demonstrations. Distortion-free excitation and polarization transfer of ∼160–170 kHz 2Hα quadrupolar coupling were presented by using a deuterium RF strength of ∼20 kHz. From these 2Hα patterns, an average backbone order parameter of S = 0.92 was determined on a deuterated SH3 sample, with an average η = 0.22. These indicate that SH3 backbone represents sizable dynamics in the microsecond timescale where the 2Hα lineshape is sensitive. Moreover, site-specific 2Hα T1 relaxation times were obtained for a proof of concept. This 3D 2HαcαNH NMR experiment has the potential to determine structure and dynamics of perdeuterated proteins by utilizing deuterium as a novel reporter.

Zwitterionic or Not? Fast and Reliable Structure Determination by Combining Crystal Structure Prediction and Solid-State NMR.

When it comes to crystal structure determination, computational approaches such as Crystal Structure Prediction (CSP) have gained more and more attention since they offer some insight on how atoms and molecules are packed in the solid state, starting from only very basic information without diffraction data. Furthermore, it is well known that the coupling of CSP with solid-state NMR (SSNMR) greatly enhances the performance and the accuracy of the predictive method, leading to the so-called CSP-NMR crystallography (CSP-NMRX). In this paper, we present the successful application of CSP-NMRX to determine the crystal structure of three structural isomers of pyridine dicarboxylic acid, namely quinolinic, dipicolinic and dinicotinic acids, which can be in a zwitterionic form, or not, in the solid state. In a first step, mono- and bidimensional SSNMR spectra, i.e., 1H Magic-Angle Spinning (MAS), 13C and 15N Cross Polarisation Magic-Angle Spinning (CPMAS), 1H Double Quantum (DQ) MAS, 1H-13C HETeronuclear CORrelation (HETCOR), were used to determine the correct molecular structure (i.e., zwitterionic or not) and the local molecular arrangement; at the end, the RMSEs between experimental and computed 1H and 13C chemical shifts allowed the selection of the correct predicted structure for each system. Interestingly, while quinolinic and dipicolinic acids are zwitterionic and non-zwitterionic, respectively, in the solid state, dinicotinic acid exhibits in its crystal structure a "zwitterionic-non-zwitterionic continuum state" in which the proton is shared between the carboxylic moiety and the pyridinic nitrogen. Very refined SSNMR experiments were carried out, i.e., 14N-1H Phase-Modulated (PM) pulse and Rotational-Echo Saturation-Pulse Double-Resonance (RESPDOR), to provide an accurate N-H distance value confirming the hybrid nature of the molecule. The CSP-NMRX method showed a remarkable match between the selected structures and the experimental ones. The correct molecular input provided by SSNMR reduced the number of CSP calculations to be performed, leading to different predicted structures, while RMSEs provided an independent parameter with respect to the computed energy for the selection of the best candidate.

Genomic and Spectroscopic Signature-Based Discovery of Natural Macrolactams.

The logical and effective discovery of macrolactams, structurally unique natural molecules with diverse biological activities, has been limited by a lack of targeted search methods. Herein, a targeted discovery method for natural macrolactams was devised by coupling genomic signature-based PCR screening of a bacterial DNA library with spectroscopic signature-based early identification of macrolactams. DNA library screening facilitated the efficient selection of 43 potential macrolactam-producing strains (3.6% of 1,188 strains screened). The PCR amplicons of the amine-deprotecting enzyme-coding genes were analyzed to predict the macrolactam type (α-methyl, α-alkyl, or β-methyl) produced by the hit strains. 1H-15N HSQC-TOCSY NMR analysis of 15N-labeled culture extracts enabled macrolactam detection and structural type assignment without any purification steps. This method identified a high-titer Micromonospora strain producing salinilactam (1), a previously reported α-methyl macrolactam, and two Streptomyces strains producing new α-alkyl and β-methyl macrolactams. Subsequent purification and spectroscopic analysis led to the structural revision of 1 and the discovery of muanlactam (2), an α-alkyl macrolactam with diene amide and tetraene chromophores, and concolactam (3), a β-methyl macrolactam with a [16,6,6]-tricyclic skeleton. Detailed genomic analysis of the strains producing 1-3 identified putative biosynthetic gene clusters and pathways. Compound 2 displayed significant cytotoxicity against various cancer cell lines (IC50 = 1.58 μM against HCT116), whereas 3 showed inhibitory activity against Staphylococcus aureus sortase A. This genomic and spectroscopic signature-based method provides an efficient search strategy for new natural macrolactams and will be generally applicable for the discovery of nitrogen-bearing natural products.

Hydrogen Atom Abstraction from an OsII(NH3)2 Complex Generates an OsIV(NH2)2 Complex: Experimental and Computational Analysis of the N-H Bond Dissociation Free Energies and Reactivity.

Double hydrogen atom abstraction from (TMP)OsII(NH3)2 (TMP = tetramesitylporphyrin) with phenoxyl or nitroxyl radicals leads to (TMP)OsIV(NH2)2. This unusual bis(amide) complex is diamagnetic and displays an N-H resonance at 12.0 ppm in its 1H NMR spectrum. 1H-15N correlation experiments identified a 15N NMR spectroscopic resonance signal at -267 ppm. Experimental reactivity studies and density functional theory calculations support relatively weak N-H bonds of 73.3 kcal/mol for (TMP)OsII(NH3)2 and 74.2 kcal/mol for (TMP)OsIII(NH3)(NH2). Cyclic voltammetry experiments provide an estimate of the pKa of [(TMP)OsIII(NH3)2]+. In the presence of Barton's base, a current enhancement is observed at the Os(III/II) couple, consistent with an ECE event. Spectroscopic experiments confirmed (TMP)OsIV(NH2)2 as the product of bulk electrolysis. Double hydrogen atom abstraction is influenced by π donation from the amides of (TMP)OsIV(NH2)2 into the d orbitals of the Os center, favoring the formation of (TMP)OsIV(NH2)2 over N-N coupling. This π donation leads to a Jahn-Teller distortion that splits the energy levels of the dxz and dyz orbitals of Os, results in a low-spin electron configuration, and leads to minimal aminyl character on the N atoms, rendering (TMP)OsIV(NH2)2 unreactive toward amide-amide coupling.

Atomic-Level Structure of Mesoporous Hexagonal Boron Nitride Determined by High-Resolution Solid-State Multinuclear Magnetic Resonance Spectroscopy and Density Functional Theory Calculations

Mesoporous hexagonal boron nitride (p-BN) has received significant attention over the last decade as a promising candidate for water cleaning/pollutant removal and hydrogen storage applications. Here, high-resolution solid-state NMR spectroscopy and plane-wave density-functional theory (DFT) calculations are used to obtain an atomic-level description of p-BN. 1H–15N or 1H–14N heteronuclear (HETCOR) correlation experiments recorded with either conventional NMR at room temperature or dynamic nuclear polarization surface-enhanced spectroscopy (DNP-SENS) at ca. 100 K reveal NB2H, NBH2, NBH3+ species residing on the edges of BN sheets. Ultra-high field 35.2 T 11B NMR spectroscopy was used to resolve 11B NMR signals from BN3, BN2Ox(OH)1–x (x = 0–1), BNOx(OH)2–x (x = 0–2), BOx(OH)3–x (x = 0–3), and BOx(OH)4–x– (x = 0–4). Importantly, 2D 11B dipolar double-quantum–single-quantum homonuclear correlation spectra reveal that many pore/defect sites are composed of boron oxide/hydroxide clusters connected to the BN framework through BN2O units. 1D and 2D 11B{15N} HETCOR NMR experiments, in addition to plane-wave DFT calculations of nine different structural models, further confirm the assignment of all NMR signals. The detailed structure determination of the pore and edge/defect sites within p-BN should further enable the rational design and development of next-generation p-BN-based materials. In addition, the techniques outlined here should be applicable to determine structure within other porous and/or boron-based materials.

The calcium-free form of atorvastatin inhibits amyloid-β(1–42) aggregation in vitro

Alzheimer's disease is characterized by the presence of extraneuronal amyloid plaques composed of amyloid-beta (Aβ) fibrillar aggregates in the brains of patients. In mouse models, it has previously been shown that atorvastatin (Ator), a cholesterol-lowering drug, has some reducing effect on the production of cerebral Aβ. A meta-analysis on humans showed moderate effects in the short term but no improvement in the Alzheimer's Disease Assessment Scale—Cognitive Subscale behavioral test. Here, we explore a potential direct effect of Ator on Aβ42 aggregation. Using NMR-based monomer consumption assays and CD spectroscopy, we observed a promoting effect of Ator in its original form (Ator-calcium) on Aβ42 aggregation, as expected because of the presence of calcium ions. The effect was reversed when applying a CaCO3-based calcium ion scavenging method, which was validated by the aforementioned methods as well as thioflavin-T fluorescence assays and transmission electron microscopy. We found that the aggregation was inhibited significantly when the concentration of calcium-free Ator exceeded that of Aβ by at least a factor of 2. The 1H–15N heteronuclear single quantum correlation and saturation-transfer difference NMR data suggest that calcium-free Ator exerts its effect through interaction with the 16KLVF19 binding site on the Aβ peptide via its aromatic rings as well as hydroxyl and methyl groups. On the other hand, molecular dynamics simulations confirmed that the increasing concentration of Ator is necessary for the inhibition of the conformational transition of Aβ from an α-helix-dominant to a β-sheet-dominant structure.

Ion bombardment technique induced alterations in the surface chemical composition and shielding parameters of polymeric material

At present, ion beam technology is one powerful technique to improve the physico-chemical properties of polymeric material. In this work, ultra high molecular weight polyethylene samples were bombarded with 1 MeV He-ion beam at fluences ranging from 1×1013 to 5×1014 ions/cm2. The changes in the chemical composition of the treated layer caused by the ion beam were investigated. The hydrogen escapes due to the ion beam irradiations were measured by the nuclear reaction analysis (NRA) technique using the 1H(15N, αγ)12C reaction. The oxygen uptake has been studied by the Rutherford backscattering spectrometry (RBS) and Fourier transform infrared spectrometer (FTIR) techniques. The partial and total mass attenuation coefficients of photons at energies 81, 356, 511 and 1332 keV were estimated using the XCOM program (version 3.1). The obtained results showed that the hydrogen release was increasing with an increase in ion fluence. Moreover, important oxygen uptake of bombarded samples was also observed. These effects led to significant improvements in the surface characteristics of the treated materials such as changes in the shielding parameters. The changes in the mass attenuation coefficients (μ/ρ) were calculated relying on the incoming photon energies and the atomic concentrations of the elements that are present in the target material. The total atomic cross-sections and effective atomic numbers were calculated by using the data of the total mass attenuation coefficients. A decrease in both the total atomic cross-section and effective atomic numbers was noticed with an increase in the ion beam fluence.

NMR 1H, 13C, 15N backbone resonance assignments of the T35S and oncogenic T35S/Q61L mutants of human KRAS4b in the active, GppNHp-bound conformation

RAS proteins cycling between the active-form (GTP-bound) and inactive-form (GDP-bound) play a key role in cell signaling pathways that control cell survival, proliferation, and differentiation. Mutations at codon 12, 13, and 61 in RAS are known to attenuate its GTPase activity favoring the RAS active state and constitutively active downstream signaling. This hyperactivation accounts for various malignancies including pancreatic, lung, and colorectal cancers. Active KRAS is found to exist in equilibrium between two rapidly interconverting conformational states (State1–State2) in solution. Due to this dynamic feature of the protein, the 1H–15N correlation cross-peak signals of several amino acid (AA) residues of KRAS belonging to the flexible loop regions are absent from its 2D 1H–15N HSQC spectrum within and near physiological solution pH. A threonine to serine mutation at position 35 (T35S) shifts the interconverting equilibrium to State1 conformation and enables the emergence of such residues in the 2D 1H–15N HSQC spectrum due to gained conformational rigidity. We report here the 1HN, 15N, and 13C backbone resonance assignments for the 19.2 kDa (AA 1–169) protein constructs of KRAS-GppNHp harboring T35S mutation (KRAST35S/C118S-GppNHp) and of its oncogenic counterpart harboring the Q61L mutation (KRAST35S/Q61L/C118S-GppNHp) using heteronuclear, multidimensional NMR spectroscopy at 298 K. High resolution NMR data allowed the unambiguous assignments of 1H–15N correlation cross-peaks for all the residues except for Met1. Furthermore, 2D 1H–15N HSQC overlay of two proteins assisted in determination of Q61L mutation-induced chemical shift perturbations for select residues in the regions of P-loop, Switch-II, and helix α3.

Spectroscopic and functional characterization of the [2Fe–2S] scaffold protein Nfu from Synechocystis PCC6803

Iron-sulfur clusters are ubiquitous cofactors required for various essential metabolic processes. Conservation of proteins required for their biosynthesis and trafficking allows for simple bacteria to be used as models to aid in exploring these complex pathways in higher organisms. Cyanobacteria are among the most investigated organisms for these processes, as they are unicellular and can survive under photoautotrophic and heterotrophic conditions. Herein, we report the potential role of Synechocystis PCC6803 NifU (now named SyNfu) as the principal scaffold protein required for iron-sulfur cluster biosynthesis in that organism. SyNfu is a well-folded protein with distinct secondary structural elements, as evidenced by circular dichroism and a well-dispersed pattern of 1H–15N HSQC NMR peaks, and readily reconstitutes as a [2Fe–2S] dimeric protein complex. Cluster exchange experiments show that glutathione can extract the cluster from holo-SyNfu, but the transfer is unidirectional. We also confirm the ability of SyNfu to transfer cluster to both human ferredoxin 1 and ferredoxin 2, while also demonstrating the capacity to deliver cluster to both monothiol glutaredoxin 3 and dithiol glutaredoxin 2. This evidence supports the hypothesis that SyNfu indeed serves as the main scaffold protein in Synechocystis, as it has been shown to be the only protein required for viability in the absence of photoautotrophic conditions. Similar to other NFU-type cluster donors and other scaffold and carrier proteins, such as ISCU, SyNfu is shown by DSC to be structurally less stable than regular protein donors, while retaining a relatively well-defined tertiary structure as represented by 1H–15N HSQC NMR experiments.