1H 15N Research Articles

A goose-type lysozyme from ostrich (Struthio camelus) egg white: multiple roles of His101 in its enzymatic reaction.

A goose-type lysozyme from ostrich egg white (OEL) was produced by Escherichia coli expression system, and the role of His101 of OEL in the enzymatic reaction was investigated by NMR spectroscopy, thermal unfolding, and theoretical modeling of the enzymatic hydrolysis of hexa-N-acetylchitohexaose, (GlcNAc)6. Although the binding of tri-N-acetylchitotriose, (GlcNAc)3, to OEL perturbed several backbone resonances in the (1)H-(15)N HSQC spectrum, the chemical shift of the backbone resonance of His101 was not significantly affected. However, apparent pKa values of His101 and Lys102 determined from the pH titration curves of the backbone chemical shifts were markedly shifted by (GlcNAc)3 binding. Thermal unfolding experiments and modeling study of (GlcNAc)6 hydrolysis using a His101-mutated OEL (H101A-OEL) revealed that the His101 mutation affected not only sugar residue affinities at subsites -3 and -2 but also the rate constant for bond cleavage. His101 appears to play multiple roles in the substrate binding and the catalytic reaction.

Nuclear magnetic resonance evidence for the dimer formation of beta amyloid peptide 1–42 in 1,1,1,3,3,3-hexafluoro-2-propanol

Alzheimer's disease involves accumulation of senile plaques in which filamentous aggregates of amyloid beta (Aβ) peptides are deposited. Recent studies demonstrate that oligomerization pathways of Aβ peptides may be complicated. To understand the mechanisms of Aβ(1–42) oligomer formation in more detail, we have established a method to produce 15N-labeled Aβ(1–42) suited for nuclear magnetic resonance (NMR) studies. For physicochemical studies, the starting protein material should be solely monomeric and all Aβ aggregates must be removed. Here, we succeeded in fractionating a “precipitation-resistant” fraction of Aβ(1–42) from an “aggregation-prone” fraction by high-performance liquid chromatography (HPLC), even from bacterially overexpressed Aβ(1–42). However, both Aβ(1–42) fractions after 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) treatment formed amyloid fibrils. This indicates that the “aggregation seed” was not completely monomerized during HFIP treatment. In addition, Aβ(1–42) dissolved in HFIP was found to display a monomer–dimer equilibrium, as shown by two-dimensional 1H–15N NMR. We demonstrated that the initial concentration of Aβ during the HFIP pretreatment altered the kinetic profiles of Aβ fibril formation in a thioflavin T fluorescence assay. The findings described here should ensure reproducible results when studying the Aβ(1–42) peptide.

Sequential protein expression and selective labeling for in-cell NMR in human cells

BackgroundIn-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein–protein interactions by NMR, only one partner should be isotopically labeled. Here we show that constitutive and transient protein expression can be combined with protein silencing to obtain selective protein labeling in human cells. MethodsWe established a human cell line stably overexpressing the copper binding protein HAH1. A second protein (human superoxide dismutase 1, SOD1) was overexpressed by transient transfection and isotopically labeled. A silencing vector containing shRNA sequences against the HAH1 gene was used to decrease the rate of HAH1 synthesis during the expression of SOD1. The levels of HAH1 mRNA and protein were measured as a function of time following transfection by RT-PCR and Western Blot, and the final cell samples were analyzed by in-cell NMR. ResultsSOD1 was ectopically expressed and labeled in a time window during which HAH1 biosynthesis was strongly decreased by shRNA, thus preventing its labeling. In-cell NMR spectra confirmed that, while both proteins were present, only SOD1 was selectively labeled and could be detected by 1H–15N heteronuclear NMR. Conclusions and general significanceWe showed that controlling protein expression by specifically silencing a stably expressed protein is a useful strategy to obtain selective isotope labeling of only one protein. This approach relies on established techniques thus permitting the investigation of protein–protein interactions by NMR in human cells.

Structural insights into mechanisms for inhibiting amyloid β42 aggregation by non-catechol-type flavonoids

The prevention of 42-mer amyloid β-protein (Aβ42) aggregation is promising for the treatment of Alzheimer’s disease. We previously described the site-specific inhibitory mechanism for Aβ42 aggregation by a catechol-type flavonoid, (+)-taxifolin, targeting Lys16,28 after its autoxidation. In contrast, non-catechol-type flavonoids (morin, datiscetin, and kaempferol) inhibited Aβ42 aggregation without targeting Lys16,28 with almost similar potencies to that of (+)-taxifolin. We herein provided structural insights into their mechanisms for inhibiting Aβ42 aggregation. Physicochemical analyses revealed that their inhibition did not require autoxidation. The 1H–15N SOFAST-HMQC NMR of Aβ42 in the presence of morin and datiscetin revealed the significant perturbation of chemical shifts of His13,14 and Gln15, which were close to the intermolecular β-sheet region, Gln15-Ala21. His13,14 also played a role in radical formation at Tyr10, thereby inducing the oxidation of Met35, which has been implicated in Aβ42 aggregation. These results suggest the direct interaction of morin and datiscetin with the Aβ42 monomer. Although only kaempferol was oxidatively-degraded during incubation, its degradation products as well as kaempferol itself suppressed Aβ42 aggregation. However, neither kaempferol nor its decomposed products perturbed the chemical shifts of the Aβ42 monomer. Aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aβ42 demonstrated that kaempferol and its degradation products inhibited the elongation rather than nucleation phase, implying that they interacted with small aggregates of Aβ42, but not with the monomer. In contrast, morin and datiscetin inhibited both phases. The position and number of hydroxyl groups on the B-ring of non-catechol-type flavonoids could be important for their inhibitory potencies and mechanisms against Aβ42 aggregation.

Convergent Synthesis Strategy of Novel Fused Azines: 1H-15N HMBC NMR as a Tool for Assertion of the Site Selectivity of Aza-Michael Addition Reaction

A unified convergent strategy has been developed for the facile synthesis of a series of novel fused azines bearing a phenothiazine moiety based on the aza-Michael addition reaction of an enaminone derivative to a variety of different heterocyclic amines under microwave irradiation conditions. The results of this study revealed that new methods are required to allow for the simple and efficient analysis of the fused azine products of this reaction other than single-crystal X-ray crystallography. With this in mind, 1H–15N HMBC experiments were used in the current study to allow for the unambiguous structural characterization of the fused azine products. These results provided further support in favor of the proposed structures and allowed for the resolution of several discrepancies reported in the literature regarding the structural characterization of these compounds. Keywords: Microwave irradiations, pyrazolo[1, 5-a]pyrimidines, triazolo[1, 5-a]pyrimidine, pyrimido[1, 2-a]benzimidazole, 1H-15N HMBC.

NMR Investigations on the Aging of Motor Oils

Aging of motor oil is characterized by diverse NMR methods to get insight into the chemical processes which occur during aging. Already one-dimensional 1H and 13C spectra reveal the relevance of chemical aging also with respect to the mechanical behavior of oils. As NMR is a quantitative analytical method, the absolute chemical changes can be observed and quantified. In addition, the spectral interpretation in terms of chemical composition is confirmed and deepened by two-dimensional 1H–1H, 1H–13C, and 1H–15N correlation spectra, as well as diffusion and relaxation experiments. The different NMR results are discussed and interpreted to obtain a thorough picture about aging processes of motor oils.

Near-surface hydrogen depletion of diamond-like carbon films produced by direct ion deposition

Amorphous atomically flat diamond-like carbon (DLC) coatings were produced by direct ion deposition using a system based on a Penning ion source, butane precursor gas and post acceleration. Hydrogen depth profiles of the DLC coatings were measured with the 15N R-NRA method using the resonant nuclear reaction 1H(15N,αγ)12C (Eres=6.385MeV). The films produced at 3.0–10.5kV acceleration voltage show two main effects. First, compared to average elemental composition of the film, the near-surface region is hydrogen depleted. The increase of the hydrogen concentration by 3% from the near-surface region towards the bulk is attributed to a growth model which favours the formation of sp2 hybridised carbon rich films in the film formation zone. Secondly, the depth at which the maximum hydrogen concentration is measured increases with acceleration voltage and is proportional to the penetration depth of protons produced by the ion source from the precursor gas. The observed effects are explained by a deposition process that takes into account the contributions of ion species, hydrogen effusion and preferential displacement of atoms during direct ion deposition.

Ion Beam Induced Modifications of Biocompatible Polymer

Ion beam bombardment has shown great potential for improving the surface properties of polymers. In this paper, the ion beam-polymer interaction mechanisms are briefly discussed. The main objective of this research was to study the effects of H-ion beam on physico-chemical properties of Ultra-high-molecular-weight polyethylene (UHMWPE) as it is frequently used in biomedical applications. UHMWPE was bombarded with 65 keV H-ions to fluences ranging from 1x1014–2x1016 ions/cm2. Changes of surface layer composition produced by ion bombardment of UHMWPE samples were studied. The hydrogen release and oxygen uptake induced by ion beam bombardment were determined by Nuclear reaction analysis (NRA) using the 1H(15N, αγ)12C and Rutherford backscattering spectrometry (RBS), respectively. Tribological and hardness properties at the polymer near surface region were studied by means of friction coefficient and micro-hardness testers, respectively. Wettability of the bombarded surfaces was determined by measuring the contact angle for distilled water. The obtained results showed that the ion bombardment induced hydrogen release increases with the increasing ion fluence. An important effect observed, was the rapid oxidation of samples, which occurs after exposure of bombarded samples to air. These effects resulted in important modifications of the surface properties of bombarded material such as change of friction coefficient, hardness and improved wettability.

A facile method for expression and purification of 15N isotope-labeled human Alzheimer’s β-amyloid peptides from E. coli for NMR-based structural analysis

Alzheimer’s disease (AD) is a progressive neurodegenerative disease affecting millions of people worldwide. AD is characterized by the presence of extracellular plaques composed of aggregated/oligomerized β-amyloid peptides with Aβ42 peptide representing a major isoform in the senile plaques. Given the pathological significance of Aβ42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of Aβ42. This report describes an efficient method to express and purify high quality 15N isotope-labeled Aβ42 for structural studies by NMR. The protocol involves utilization of an auto induction system with 15N isotope labeled medium, for high-level expression of Aβ42 as a fusion with IFABP. After the over-expression of the 15N isotope-labeled IFABP-Aβ42 fusion protein in the inclusion bodies, pure 15N isotope-labeled Aβ42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled Aβ42 peptide (Garai et al., 2009). We obtain a final yield of ∼6mg/L culture for 15N isotope-labeled Aβ42 peptide. Mass spectrometry and 1H–15N HSQC spectra of monomeric Aβ42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with 15N and 13C in Aβ42 peptide as well as its other variants including any Aβ42 peptide mutants.

Strong in Vitro Cytotoxic Potential of New Ruthenium–Cymene Complexes

Two p-cymenerutheniumchlorido complexes with thiourea derivative of 7-chloroquinoline (C1) and pyridine-3-imidazole (C2) were synthesized starting from [(η6-p-cymene)RuCl2]2 and corresponding ligands. The structures of complexes were determined with elemental analysis and IR, ESIMS, 1H and 13C{1H} NMR, and 2D 1H–15N correlation NMR spectroscopy. Cytotoxic activities examined by the MTT assay were performed in five human neoplastic cell lines (HeLa, K562, A549, MDA-MB-231, EA.hy926) and one nontumor human fetal lung fibroblast cell line (MRC-5). Tested complexes exhibited low micromolar activities with IC50 in the range 11.03–56.45 μM, while ligands L1 and L2 were significantly less active. Complex C1 showed cytoselective activity toward the K562 cell line (IC50 = 11.03 ± 1.39 μM) and was 3 times less active against the nontumor MRC-5 cell line. Flow cytometry analysis indicated that complexes C1 and C2 after 24 h treatment caused a concentration-dependent increase of the apoptotic sub-G1 fraction (up to 1...

Correction to On the Analytical Superiority of 1D NMR for Fingerprinting the Higher Order Structure of Protein Therapeutics Compared to Multidimensional NMR Methods

An important aspect in the analytical characterization of protein therapeutics is the comprehensive characterization of higher order structure (HOS). Nuclear magnetic resonance (NMR) is arguably the most sensitive method for fingerprinting HOS of a protein in solution. Traditionally, 1H–15N or 1H–13C correlation spectra are used as a “structural fingerprint” of HOS. Here, we demonstrate that protein fingerprint by line shape enhancement (PROFILE), a 1D 1H NMR spectroscopy fingerprinting approach, is superior to traditional two-dimensional methods using monoclonal antibody samples and a heavily glycosylated protein therapeutic (Epoetin Alfa). PROFILE generates a high resolution structural fingerprint of a therapeutic protein in a fraction of the time required for a 2D NMR experiment. The cross-correlation analysis of PROFILE spectra allows one to distinguish contributions from HOS vs protein heterogeneity, which is difficult to accomplish by 2D NMR. We demonstrate that the major analytical limitation of tw...

Zinc Ion-binding Activity of an Anti-ZnO VHH Antibody, 4F2

Abstract The interaction between biomolecules and inorganic materials has gained much interest for the development of sensors and highly controlled materials in scale and composition. For this purpose, 4F2, a VHH antibody with high affinity and specificity toward zinc oxide (ZnO) surfaces has been constructed. Here, we report that 4F2 also has an affinity toward zinc ion (Zn2+). The stability of 4F2 and its Zn2+ complexes was determined using differential scanning calorimetry. We then determined the binding affinity between 4F2 and Zn2+ by using isothermal calorimetry. To further elucidate the molecular binding mechanism of 4F2 toward Zn2+, we applied the 1H–15N heteronuclear single quantum coherence NMR technique. We found that 4F2 recognized Zn2+ near the complementarity determining region 3 (CDR3). Our results provide the basis for the recognition mechanism of ZnO by 4F2.

Extending long-range heteronuclear NMR connectivities by HSQMBC-COSY and HSQMBC-TOCSY experiments

The detection of long-range heteronuclear correlations presenting J(CH) coupling values smaller than 1–2Hz is a challenge in the structural analysis of small molecules and natural products. HSQMBC-COSY and HSQMBC-TOCSY pulse schemes are evaluated as complementary NMR methods to standard HMBC/HSQMBC experiments. Incorporation of an additional J(HH) transfer step in the basic HSQMBC pulse scheme can favor the sensitive observation of traditionally missing or very weak correlations and, in addition, facilitates the detection of a significant number of still longer-range connectivities to both protonated and non-protonated carbons under optimum sensitivity conditions. A comparative 1H–13C study is performed using strychnine as a model compound and several examples are also provided including 1H–15N applications.

Studies of Stereochemistry of 3-(Arylhydrazono)Furan-2(3Н)-Ones, Synthesis of 4-(Arylhydrazono)Pyridazin-3(1Н)-Ones

The structural characteristics of 5-aryl-3-(arylhydrazono)furan-2(3Н)-ones accessed via azocoupling of furan-2(3Н)-ones with aryldiazonium salts were established by NMR spectroscopy (including two-dimensional NMR experiments 1H–13C HMQC, 1H–15N HMQC, 1H–13C HMBC, NOE) and X-ray structural analysis techniques. The reaction of synthesized compounds with hydrazine leading to the formation of six-membered heterocycles, hydrazono-substituted 4,5-dihydropyridazin-3(2Н)-ones, was studied.

An optimized method for (15)N R(1) relaxation rate measurements in non-deuterated proteins.

15N longitudinal relaxation rates are extensively used for the characterization of protein dynamics; however, their accurate measurement is hindered by systematic errors. 15N CSA/1H–15N dipolar cross-correlated relaxation (CC) and amide proton exchange saturation transfer from water protons are the two main sources of systematic errors in the determination of 15N R1 rates through 1H–15N HSQC-based experiments. CC is usually suppressed through a train of 180° proton pulses applied during the variable 15N relaxation period (T), which can perturb water magnetization. Thus CC cancellation is required in such a way as to minimize water saturation effects. Here we examined the level of water saturation during the T period caused by various types of inversion proton pulses to suppress CC: (I) amide-selective IBURP-2; (II) cosine-modulated IBURP-2; (III) Watergate-like blocks; and (IV) non-selective hard. We additionally demonstrate the effect of uncontrolled saturation of aliphatic protons on 15N R1 rates. In this paper we present an optimized pulse sequence that takes into account the crucial effect of controlling also the saturation of the aliphatic protons during 15N R1 measurements in non-deuterated proteins. We show that using cosine-modulated IBURP-2 pulses spaced 40 ms to cancel CC in this optimized pulse program is the method of choice to minimize systematic errors coming from water and aliphatic protons saturation effects.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-015-9937-4) contains supplementary material, which is available to authorized users.

Bacterial expression of the phosphodiester-binding site of the cation-independent mannose 6-phosphate receptor for crystallographic and NMR studies

The cation-independent mannose 6-phosphate receptor (CI-MPR) is a multifunctional protein that interacts with diverse ligands and plays central roles in autophagy, development, and tumor suppression. By delivering newly synthesized phosphomannosyl-containing acid hydrolases from the Golgi to endosomal compartments, CI-MPR is an essential component in the generation of lysosomes that are critical for the maintenance of cellular homeostasis. The ability of CI-MPR to interact with ∼60 different acid hydrolases is facilitated by its large extracellular region, with four out of its 15 domains binding phosphomannosyl residues. Although the glycan specificity of CI-MPR has been elucidated, the molecular basis of carbohydrate binding has not been determined for two out of these four carbohydrate recognition domains (CRD). Here we report expression of CI-MPR’s CRD located in domain 5 that preferentially binds phosphodiester-containing glycans. Domain 5 of CI-MPR was expressed in Escherichia coli BL21 (DE3) cells as a fusion protein containing an N-terminal histidine tag and the small ubiquitin-like modifier (SUMO) protein. The His6-SUMO-CRD construct was recovered from inclusion bodies, refolded in buffer to facilitate disulfide bond formation, and subjected to Ni-NTA affinity chromatography and size exclusion chromatography. Surface plasmon resonance analyses demonstrated that the purified protein was active and bound phosphorylated glycans. Characterization by NMR spectroscopy revealed high quality 1H–15N HSQC spectra. Additionally, crystallization conditions were identified and a crystallographic data set of the CRD was collected to 1.8Å resolution. Together, these studies demonstrate the feasibility of producing CI-MPR’s CRD suitable for three-dimensional structure determination by NMR spectroscopic and X-ray crystallographic approaches.

Extraction of chitosan from Aspergillus niger mycelium and synthesis of hydrogels for controlled release of betahistine

Chitosan was extracted from the fungus Aspergillus niger, an alternative source of chitin that is widely available as a byproduct of the industrial production of citric acid. Chitosan with deacetylation degree (DD) of 73.6% was characterized by elemental analysis, capillary viscometry (molecular weight of 1.9×105g/mol), Fourier transform infrared (FTIR), nuclear magnetic resonance (1H NMR, 13C NMR and 15N NMR) spectroscopies, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Fungal chitosan was crosslinked with glutaraldehyde and glutaric acid to obtain hydrogels. Chitosan hydrogels were characterized by FTIR and by scanning electron microscopy (SEM), which showed that these materials have irregular, polydisperse, and interconnected pores. Kinetic studies of the release of betahistine from the swollen hydrogels showed a Fickian diffusion mechanism. Finally, hydrolytic degradation of chitosan hydrogels under simulated physiological conditions (pH 7.4 and 37°C) was investigated as well as in vivo biocompatibility tests using New Zealand white rabbits as animal models.

Fragment Screening and Druggability Assessment for the CBP/p300 KIX Domain through Protein-Observed19F NMR Spectroscopy

19F NMR spectroscopy of labeled proteins is a sensitive method for characterizing structure, conformational dynamics, higher-order assembly, and ligand binding. Fluorination of aromatic side chains has been suggested as a labeling strategy for small-molecule ligand discovery for protein–protein interaction interfaces. Using a model transcription factor binding domain of the CREB binding protein (CBP)/p300, KIX, we report the first full small-molecule screen using protein-observed 19F NMR spectroscopy. Screening of 508 compounds and validation by 1H–15N HSQC NMR spectroscopy led to the identification of a minimal pharmacaphore for the MLL-KIX interaction site. Hit rate analysis for the CREB-KIX and MLL-KIX sites provided a metric to assess the ligandability or “druggability” of each interface informing future medicinal chemistry efforts. The structural information from the simplified spectra and data collection speed, affords a new screening tool for analysis of protein interfaces and discovery of small molecules.

ChemInform Abstract: Hydrogen Detection Near Surfaces and Shallow Interfaces with Resonant Nuclear Reaction Analysis

AbstractReview: 783 refs.

Functional Siglec lectin domains from soluble expression in the cytoplasm of Escherichia coli

Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of mammalian cell-surface receptors that are involved in cell–cell interactions and signaling functions, primarily expressed on cells of the immune system. Key to their function is their specific binding of distinct sialylated glycan ligands mediated via an N-terminal carbohydrate recognition (lectin) domain. Studies concerning the molecular basis of their individual carbohydrate specificities are rare due to the absence of suitable recombinant expression methods for producing these disulfide-containing proteins in sufficient quantities required for their in-depth in vitro characterization. We established an efficient E. coli-based expression and purification method for Siglec lectin domains, utilizing the trxB gor suppressor strain Rosetta-gami B (DE3) in which proper folding with intact disulfide bonds was achieved in the cytoplasm. The approach is demonstrated for human Siglec-7, -8 and -9 lectin domains and works equally well for expression in nutrient-rich (LB) or minimal growth medium, allowing stable-isotope labeling for NMR studies. The recombinant proteins were properly folded as proven by 2D 1H–15N HSQC NMR spectroscopy and by thermal unfolding followed by CD spectroscopy, and functionally active as confirmed by monitoring ligand binding using NMR titration experiments. Our method enables efficient production of homogeneous and active protein samples in milligram quantities. Its implementation will significantly enhance future structure–function studies of this important class of immune-modulating receptors and will support a variety of applications including screening for natural and synthetic ligands or the development of fluorescently-labeled molecular tools for glycan ligand detection or flow-cytometric cell sorting.