NMR Resonances Research Articles

1HN, 13C, and 15N backbone resonance assignments of the SET/TAF-1β/I2PP2A oncoprotein (residues 23-225).

SET (TAF-1β/I2PP2A) is a ubiquitously expressed, multifunctional protein that plays a role in regulating diverse cellular processes, including cell cycle progression, migration, apoptosis, transcription, and DNA repair. SET expression is ubiquitous across all cell types. However, it is overexpressed or post-translationally modified in several solid tumors and blood cancers, where expression levels are correlated with worsening clinical outcomes.SET exerts its oncogenic effects primarily through the formation of antagonistic protein complexes with the tumor suppressor, protein phosphatase 2A (PP2A), and the well-known metastasis suppressor, nm23-H1. PP2A inhibition is often observed as a secondary driver of tumorigenesis and metastasis in human cancers. Preclinical studies have shown that the pharmacological reactivation of PP2A combined with potent inhibitors of the primary driver oncogene produces synergistic cell death and decreased drug resistance.Therefore, the development of novel inhibitors of the SET-PP2A interaction presents an attractive approach to reactivation of PP2A, and thereby, tumor suppression. NMR provides a unique platform to investigate protein targets in their natively folded state to identify protein and small-molecule ligands and report on the protein internal dynamics.The backbone 1HN, 13C, and 15N NMR resonance assignments were completed for the 204 amino acid nucleosome assembly protein-1 (NAP-1) domain of the human SET oncoprotein (residues 23-225).These assignments provide a vital first step toward the development of novel PP2A reactivators via SET-selective inhibition.

NMR resonance assignments of the PR-10 allergens Act c 8 and Act d 8 from golden and green kiwifruit

Kiwifruits have become one of the most common food sources triggering allergic reactions. In patients suffering from birch pollen related food allergy, reactions result from initial sensitization to the birch (Betula verrucosa) pollen allergen Bet v 1, followed by immunological cross-reactivity to structurally homologous proteins in kiwifruit. Clinical symptoms range from scratching and itching of the oral cavity to more severe immunological reactions such as rhino conjunctivitis. In this work we assigned backbone and side chain 1H, 13C and 15N chemical shifts of the 17 kDa PR-10 allergens Act c 8.0101 and Act d 8.0101 from golden (Actinidia chinesis) and green (Actinidia deliciosa) kiwifruit by solution NMR spectroscopy. The chemical shift data confirm the characteristic Bet v 1 fold for both proteins, consisting of a seven-stranded antiparallel β-sheet interrupted by two short α-helices, along with a long C-terminal α-helix. Our data provide the basis for determining the three-dimensional solution structures of these proteins and characterizing their immunological cross-reactivity on a structural basis.

The Incorporation of Labile Protons into Multidimensional NMR Analyses: Glycan Structures Revisited

Glycan structures are often stabilized by a repertoire of hydrogen-bonded donor/acceptor groups, revealing longer-lived structures that could represent biologically relevant conformations. NMR provides unique data on these hydrogen-bonded networks from multidimensional experiments detecting cross-peaks resulting from through-bond (TOCSY) or through-space (NOESY) interactions. However, fast OH/H2O exchange, and the spectral proximity among these NMR resonances, hamper the use of glycans’ labile protons in such analyses; consequently, studies are often restricted to aprotic solvents or supercooled aqueous solutions. These nonphysiological conditions may lead to unrepresentative structures or to probing a small subset of accessible conformations that may miss “active” glycan conformations. Looped, projected spectroscopy (L-PROSY) has been recently shown to substantially enhance protein NOESY and TOCSY cross-peaks, for 1Hs that undergo fast exchange with water. This study shows that even larger enhancements can be obtained for rapidly exchanging OHs in saccharides, leading to the retrieval of previously undetectable 2D TOCSY/NOESY cross-peaks with nonlabile protons. After demonstrating ≥300% signal enhancements on model monosaccharides, these experiments were applied at 1 GHz to elucidate the structural network adopted by a sialic acid homotetramer, used as a model for α,2–8 linked polysaccharides. High-field L-PROSY NMR enabled these studies at higher temperatures and provided insight previously unavailable from lower-field NMR investigations on supercooled samples, involving mostly nonlabile nuclei. Using L-PROSY’s NOEs and other restraints, a revised structural model for the homotetramer was obtained combining rigid motifs and flexible segments, that is well represented by conformations derived from 40 μs molecular dynamics simulations.

Anomalous magnetism of uranium(IV)-oxo and -imido complexes reveals unusual doubly degenerate electronic ground states

Anomalous magnetism of uranium(IV)-oxo and -imido complexes reveals unusual doubly degenerate electronic ground states

NHC-Stabilized 1,2-Dihalodiborenes: Synthesis, Characterization, and Reactivity Toward Elemental Chalcogens.

The 2-fold reduction of B2X4(NHC)2 (X = Cl, Br, I; NHC = (un)saturated N-heterocyclic carbene) yields the corresponding green-colored 1,2-dihalodiborenes B2X2(NHC)2, the 11B NMR resonances of which are strongly upfield-shifted upon descending the halide group. The diborenes crystallize as the trans isomers, with relatively short B═B double bonds (1.513(9) to 1.568(4) Å). Cyclic voltammetric experiments with these diborenes reveal reversible one-electron oxidation processes to the corresponding diboron radical cation (E1/2 = -1.16 to -1.50 V); the reducing power of B2X2(NHC)2 increasing with the electronegativity of the halide and for the less π-accepting unsaturated NHCs. The main UV-vis absorption (393-463 nm), which corresponds mainly to a highest occupied molecular orbital (HOMO) → lowest unoccupied molecular orbital (LUMO) transition, undergoes a blueshift upon descending the halide group and shows some dependence on the stereoelectronics of the NHC ligands. Computational analyses show that the HOMO of B2X2(NHC)2 is mostly localized on the B═B π bond, with the contribution from halide p orbitals decreasing down the group, and the saturated NHCs affording some π-bonding delocalization over the B-CNHC bonds. The calculated HOMO and LUMO energies decrease upon descending the halide group, while the HOMO-LUMO gap also decreases, correlating well with the cyclovoltammetry and UV-vis data. The reactions of B2Br2(NHC)2 with elemental sulfur and red selenium lead to the formation of the corresponding diborathiiranes and seleniranes, respectively, which were characterized by NMR and UV-vis spectroscopy, cyclic voltammetry, and X-ray diffraction analyses. In one case, an additional one-electron oxidation yields a unique cyclic B2Se radical cation. Computational analyses show that the localization of the HOMO and HOMO - 1 of the diboraseleniranes is inverted compared to the diborathiiranes.

Protein yoga: Conformational versatility of the Hemolysin II C-terminal domain detailed by NMR structures for multiple states.

The C-terminal domain of Bacillus cereus hemolysin II (HlyIIC), stabilizes the trans-membrane-pore formed by the HlyII toxin and may aid in target cell recognition. Initial efforts to determine the NMR structure of HlyIIC were hampered by cis/trans isomerization about the single proline at position 405 that leads to doubling of NMR resonances. We used the mutant P405M-HlyIIC that eliminates the cis proline to determine the NMR structure of the domain, which revealed a novel fold. Here, we extend earlier studies to the NMR structure determination of the cis and trans states of WT-HlyIIC that exist simultaneously in solution. The primary structural differences between the cis and trans states are in the loop that contains P405, and structurally adjacent loops. Thermodynamic linkage analysis shows that at 25 C the cis proline, which already has a large fraction of 20% in the unfolded protein, increases to 50% in the folded state due to coupling with the global stability of the domain. The P405M or P405A substitutions eliminate heterogeneity due to proline isomerization but lead to the formation of a new dimeric species. The NMR structure of the dimer shows that it is formed through domain-swapping of strand β5, the last segment of secondary structure following P405. The presence of P405 in WT-HlyIIC strongly disfavors the dimer compared to the P405M-HlyIIC or P405A-HlyIIC mutants. The WT proline may thus act as a "gatekeeper," warding off aggregative misfolding.

NMR resonance assignments for the nucleotide binding domains of the E. coli clamp loader complex γ subunit.

The E. coli γ clamp loader is a pentameric complex of δ, δ' and three γ subunits that opens and loads β-clamp proteins onto DNA in an ATP-dependent process essential for efficient DNA replication. ATP binding to the γ subunits promotes conformational changes that enable the clamp loader to bind and open the ring-shaped β-clamp homodimer. Here we report the nearly complete backbone and side-chain 1H, 13C and 15N NMR resonance assignments of the 242-residue truncated γ subunit of the clamp loader complex, which includes the N-terminal mini (domain I) and lid (domain II) domains. This construct represents the nucleotide binding module in the clamp loader complex and provides a model system for studies of conformational rearrangements of the clamp loader induced by nucleotide binding.

Nature of Heterobinuclear Ni(I) Complexes Formed upon the Activation of the α-Diimine Complex LNiIIBr2 with AlMe3 and MMAO

The nature of Ni(I) species formed upon the activation of the N,N-α-diimine Ni(II) ethylene polymerization precatalyst LNiIIBr2 (1) with AlMe3 and MMAO has been studied in detail by 1H, 2H NMR, and electron paramagnetic resonance (EPR) spectroscopy (L = 1,4-bis(2,6-diisopropylphenyl)-2,3-dimethyl-1,4-diazabuta-1,3-diene). It has been shown that neutral paramagnetic NMR and EPR active heterobinuclear complexes of types LNiI(μ-Me)(μ-Br)AlR2 and LNiI(μ-Me)2AlR2 (R = Me in the case of AlMe3 and R = Me or iBu in the case of MMAO) predominate in toluene solutions of 1/AlMe3 and 1/MMAO at room temperature. The effect of the cocatalyst/catalyst ratio on the relative concentrations of LNiI(μ-Me)(μ-Br)AlR2 and LNiI(μ-Me)2AlR2 has been studied and their role in ethylene polymerization is discussed.

Effect of vacuum impregnated fish gelatin and grape seed extract on moisture state, microbiota composition, and quality of chilled seabass fillets

The effect of fish gelatin (FG) and grape seed extract (GSE) on microbiota composition and moisture state of fish was unexplored. Herein, this study aimed to evaluate the single and combined (FGG) effects on seabass during storage (4 °C) with assistant of vacuum impregnation and to elucidate the underlying preservative mechanism. As suggested by low-field NMR and magnetic resonance imaging, FGG-treated seabass presented higher water holding capacity by controlling transformation from immobilised to free water. Moreover, the total viable count and spoilage bacteria were reduced by > 1 log CFU/g as compared to the control. Changes in microbial flora analysed using high throughput sequencing further indicated that GSE contributed to the notably suppressed growth of Pseudomonas. Also, the accumulation of biogenic amines especially putrescine was decreased (over 0.5-fold) under the combination treatment as compared to the control (P < 0.05). The results suggest that FGG is promising for seabass preservation.

The effects of sulphur dioxide on wine metabolites: New insights from 1H NMR spectroscopy based in-situ screening, detection, identification and quantification

The wine industry routinely employs sulphur dioxide (SO2) as an additive for antiseptic, antioxidasic and antioxidant activities. Some of the most important wine metabolites such as acetaldehyde, diacetyl, pyruvic acid, α-ketoglutaric acid, hydroxymethylfurfural, furfural and acetoin are known to react with SO2 over time to form adducts. 1H NMR spectroscopy of wine can be tricky as metabolites exist either in original form or as O-adducts or as S-adducts at different SO2 concentrations. We are proposing a methodology for metabolites identification including all the possible SO2 generated species. 1H NMR spectroscopy is able to highlight the structural changes that a metabolite undergoes in wine in the presence of SO2. The 1H NMR spectra of the main wine metabolites known to react with SO2 were recorded in wine-model solutions at SO2 concentrations at 0, 50, 100 and 150 mg/L that are all below the maximum level allowed by the European Union regulations. Acetaldehyde was completely transformed into the corresponding hydroxysulfonate at a SO2 concentration of 100 mg/L. The concentration of this metabolite was assayed in ten commercial Moscato wine samples including the 1H NMR resonances of all its identified forms. The results corresponded to those determined according to the OIV standard protocols.

Assignments of 19F NMR resonances and exploration of dynamics in a long-chain flavodoxin

Flavodoxin is a small protein that employs a non-covalently bound flavin to mediate single-electron transfer at low potentials. The long-chain flavodoxins possess a long surface loop that is proposed to interact with partner proteins. We have incorporated 19F-labeled tyrosine in long-chain flavodoxin from Rhodopseudomonas palustris to gain a probe of possible loop dynamics, exploiting the presence of a Tyr in the long loop in addition to Tyr residues near the flavin. We report 19F resonance assignments for all four Tyrs, and demonstration of a pair of resonances in slow exchange, both corresponding to a Tyr adjacent to the flavin. We also provide evidence for dynamics affecting the Tyr in the long loop. Thus, we show that 19F NMR of 19F-Tyr labeled flavodoxin holds promise for monitoring possible changes in conformation upon binding to partner proteins.

NMR and EPR reveal a compaction of the RNA-binding protein FUS upon droplet formation.

Many RNA-binding proteins undergo liquid-liquid phase separation, which underlies the formation of membraneless organelles, such as stress granules and P-bodies. Studies of the molecular mechanism of phase separation in vitro are hampered by the coalescence and sedimentation of organelle-sized droplets interacting with glass surfaces. Here, we demonstrate that liquid droplets of fused in sarcoma (FUS)-a protein found in cytoplasmic aggregates of amyotrophic lateral sclerosis and frontotemporal dementia patients-can be stabilized in vitro using an agarose hydrogel that acts as a cytoskeleton mimic. This allows their spectroscopic characterization by liquid-phase NMR and electron paramagnetic resonance spectroscopy. Protein signals from both dispersed and condensed phases can be observed simultaneously, and their respective proportions can be quantified precisely. Furthermore, the agarose hydrogel acts as a cryoprotectant during shock-freezing, which facilitates pulsed electron paramagnetic resonance measurements at cryogenic temperatures. Surprisingly, double electron-electron resonance measurements revealed a compaction of FUS in the condensed phase.

NMR resonance assignments for a docking domain pair with an attached thiolation domain from the PAX peptide-producing NRPS from Xenorhabdus cabanillasii

Non-ribosomal peptide synthetases (NRPSs) are large multienzyme machineries. They synthesize numerous important natural products starting from amino acids. For peptide synthesis functionally specialized NRPS modules interact in a defined manner. Individual modules are either located on a single or on multiple different polypeptide chains. The “peptide-antimicrobial-Xenorhabdus” (PAX) peptide producing NRPS PaxS from Xenorhabdus bacteria consists of the three proteins PaxA, PaxB and PaxC. Different docking domains (DDs) located at the N-termini of PaxB and PaxC and at the C-termini of PaxA and BaxB mediate specific non-covalent interactions between them. The N-terminal docking domains precede condensation domains while the C-terminal docking domains follow thiolation domains. The binding specificity of individual DDs is important for the correct assembly of multi-protein NRPS systems. In many multi-protein NRPS systems the docking domains are sufficient to mediate the necessary interactions between individual protein chains. However, it remains unclear if this is a general feature for all types of structurally different docking domains or if the neighboring domains in some cases support the function of the docking domains. Here, we report the 1H, 13C and 15 N NMR resonance assignments for a C-terminal di-domain construct containing a thiolation (T) domain followed by a C-terminal docking domain (CDD) from PaxA and for its binding partner – the N-terminal docking domain (NDD) from PaxB from the Gram-negative entomopathogenic bacterium Xenorhabdus cabanillasii JM26 in their free states and for a 1:1 complex formed by the two proteins. These NMR resonance assignments will facilitate further structural and dynamic studies of this protein complex.

Structural insights into α-synuclein monomer–fibril interactions

Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson's disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.

A method for increasing the solubility of industrial azo pigments for 13 C and 1 H NMR spectra and the assignments of their 13 C and 1 H NMR peaks.

A method for increasing the solubilities of industrial azo pigments in DMSO by adding DBU (1,8-diaza-7-bicyclo[5.4.0]undecene) has been developed. This facilitated the acquisition of solution 13 C NMR spectra of the pigments. This method was applied to four types of azo pigments: naphthol AS (3-hydroxy-2-naphthoic acid anilide) pigments, naphthol pigments, pyrazolone pigments and acetoacetanilide pigments. This represents the first solution 13 C NMR spectra for naphthol AS pigments. Altogether 18 industrial azo pigments were analysed using 1D and 2D NMR techniques. The proton and corresponding carbon NMR resonances of these pigments have all been assigned.

Backbone-independent NMR resonance assignments of methyl probes in large proteins

Methyl-specific isotope labeling is a powerful tool to study the structure, dynamics and interactions of large proteins and protein complexes by solution-state NMR. However, widespread applications of this methodology have been limited by challenges in obtaining confident resonance assignments. Here, we present Methyl Assignments Using Satisfiability (MAUS), leveraging Nuclear Overhauser Effect cross-peak data, peak residue type classification and a known 3D structure or structural model to provide robust resonance assignments consistent with all the experimental inputs. Using data recorded for targets with known assignments in the 10–45 kDa size range, MAUS outperforms existing methods by up to 25,000 times in speed while maintaining 100% accuracy. We derive de novo assignments for multiple Cas9 nuclease domains, demonstrating that the methyl resonances of multi-domain proteins can be assigned accurately in a matter of days, while reducing biases introduced by manual pre-processing of the raw NOE data. MAUS is available through an online web-server.

Deciphering molecular mechanics in the taste masking ability of Maltodextrin: Developing pediatric formulation of Oseltamivir for viral pandemia

Present research work was aimed at masking the bitter taste of anti- viral drug Oseltamivir phosphate (Ost) by complexing it with pea starch maltodextrin- Kleptose Linecaps® (Mld). The Ost groups involved in triggering the bitter sensation were identified by computationally assessing its interaction with human bitter taste receptor hTAS2R 38. A series of exhaustive molecular dynamics (MD) simulation was run using Schrodinger® suite to understand the type of interaction of Ost with Mld. Experimentally, complexes of Ost with Mld were realized by solution method. The complexes were characterized using differential scanning colorimetry (DSC), fourier transform-infrared spectroscopy (FT-IR), powder X-ray diffraction (PXRD), hot stage microscopy (HSM), scanning electron microscopy (SEM), proton NMR (1H-NMR) and Carbon-13 nuclear magnetic resonance (13C-NMR). Ost-oral dispersible mini tablets (ODMT) were prepared by direct compression and optimised using mixture designs. Finally, bitter taste perception of Ost-ODMT was evaluated in healthy human volunteers of either sex. Computational assessment, involving interaction of Ost with bitter receptor, predicted the involvement of free amino group of Ost in triggering the bitter response whereas, MD simulation predicted the formation of stable complex between Ost and double helical confirmation of Mld. Different characterization techniques confirmed the findings of MD simulation. Results from the taste assessment in human volunteers revealed a significant reduction in bitter taste of prepared Ost-ODMT

Исследование ледовых композиционных материалов, армированных нанодисперсными волокнами оксида алюминия, методами ядерного магнитного резонанса

The aqueous suspensions of nanosized alumina fibers and ice composite materials based on them were studied using Pulsed-Field Gradient NMR and Magnetic Resonance Imaging methods. It is shown that the introduction of nanofibers does not lead to noticeable structural effects in suspensions in the concentration range of 1 - 10 wt. % Al2O3. It was found that a high concentration of filler significantly changes the morphology and texture of the ice matrix in composites: it becomes more homogeneous, with a high degree of continuity; when the melting front passes through, internal regions of thawing or failure of the integrity of the composite are not visualized. Yet the introduction of nanofibers into ice composites has a weak effect on the quantitative dynamics of heat transfer processes, making the rate of freezing/thawing front propagation to be similar in different samples at comparable temperatures of the process.

Environment and coordination of FeMo-co in the nitrogenase metallochaperone NafY.

In nitrogenase biosynthesis, the iron-molybdenum cofactor (FeMo–co) is externally assembled at scaffold proteins and delivered to the NifDK nitrogenase component by the NafY metallochaperone. Here we have used nuclear magnetic resonance, molecular dynamics, and functional analysis to elucidate the environment and coordination of FeMo–co in NafY. H121 stands as the key FeMo–co ligand. Regions near FeMo–co diverge from H121 and include the η1, α1, α2 helical lobe and a narrow path between H121 and C196.

Gold(I) ethylene complexes supported by electron-rich scorpionates.

Ethylene complexes of gold(i) have been stabilized by electron-rich, κ2-bound tris(pyrazolyl)borate ligands. Large up-field shifts of olefinic carbon NMR resonances and relatively long C[double bond, length as m-dash]C distances of gold bound ethylene are indicative of significant Au(i) → ethylene π-backbonding relative to the analog supported by a weakly donating ligand, consistent with the computational data.