Heteronuclear Single Quantum Correlation NMR Research Articles

The Development of a High-Throughput Homonuclear Decoupling HSQC NMR Platform for the Determination of 10 Sex Hormones in Animal-Source Food and Medicines

Owing to their endocrine disruption effect, the hormone levels in animal-source food and medicines need to be efficiently and accurately quantified by a reliable analytical method. In the current study, by using a homonuclear decoupling and heteronuclear single quantum correlation (HSQC) experiment, coupled with non-uniform sampling (NUS) that was used to shorten the experimental time, we developed a method to quantify 10 hormone residues in animal-source products. This method was validated following the guidelines of USP–NF 2022. The application of the homonuclear decoupling (HD) technique to conventional HSQC yielded 2D spectra that exhibited excellent signal separation and specificity. For all the tested hormones, good linearity with correlation coefficients of more than 0.99 was observed in the linear range of 0.2–6 mg/0.6 mL. Satisfactory precision and recoveries of spiked animal samples were also obtained. Finally, the method was applied in residue determination of 10 hormones in real animal-source samples at the ug/g level.

Profiling Enzyme Activity of l-Asparaginase II by NMR-Based Methyl Fingerprinting at Natural Abundance.

l-asparaginase II (MW 135 kDa) from E. coli is an FDA-approved protein drug used for the treatment of childhood leukemia. Despite its long history as a chemotherapeutic, the structural basis of enzyme action, in solution, remains widely contested. In this work, methyl-based 2D [1H-13C]-heteronuclear single-quantum correlation (HSQC) NMR, at natural abundance, has been used to profile the enzymatic activity of the commercially available enzyme drug. The [1H-13C]-HSQC NMR spectra of the protein reveal the role of a flexible loop segment in the activity of the enzyme, in solution. Addition of asparagine to the protein results in distinct conformational changes of the loop that could be signatures of intermediates formed in the catalytic reaction. To this end, an isothermal titration calorimetry (ITC)-based assay has been developed to measure the enzymatic reaction enthalpy, as a marker for its activity. Combining both ITC and NMR, it was shown that the disruption of the protein conformation can result in the loss of function. The scope, robustness, and validity of the loop fingerprints in relation to enzyme activity have been tested under different solution conditions. Overall, our results indicate that 2D NMR can be used reliably to gauge the structure-function of this enzyme, bypassing the need to label the protein. Such natural abundant NMR methods can be potentially extended to probe the structure-function aspects of high-molecular-weight protein therapeutics (glycosylated protein drugs, enzymes, therapeutic monoclonal antibodies, antibody-drug conjugates, and Fc-fusion proteins), where (a) flexible loops are required for their function and (b) isotope labeling may not be straightforward.

NMR Study on Laccase Polymerization of Kraft Lignin Using Different Enzymes Source.

The usage of laccases is a sustainable and environmentally friendly approach to modifying the Kraft lignin structure for use in certain applications. However, the inherent structure of Kraft lignin, as well as that resulting from laccase modification, still presents challenges for fundamental comprehension and successful lignin valorization. In this study, bacterial and fungal laccases were employed to modify eucalypt Kraft lignin. To evaluate the type and range of the chemical and structural changes of laccase-treated lignins, different NMR techniques, including solution 1H and 2D NMR (heteronuclear single quantum correlation (HSQC)), and solid-state 13C NMR, were applied. Size exclusion chromatography and infrared spectroscopy were also used. Interestingly, HSQC analysis showed substantial changes in the oxygenated aliphatic region of lignins, showing an almost complete absence of signals corresponding to side-chains due to laccase depolymerization. Simultaneously, a significant loss of aromatic signals was observed by HSQC and 1H NMR, which was attributed to a deprotonation of the lignin benzenic rings due to polymerization/condensation by laccase reactions. Then, condensed structures, such as α-5', 5-5', and 4-O-5', were detected by HSQC and 13C NMR, supporting the increment in molecular weight, as well as the phenolic content reduction determined in lignins.

Gelation of konjac glucomannan by acetylmannan esterases from Aspergillus oryzae

Konjac glucomannan (KGM) is a principal component of the gelatinous food Konjac. Konjac production through alkali treatment releases an undesirable amine-odor. Two acetylesterases (AME1 and AME2) active against konjac glucomannan (polymer or oligomer) were purified from the supernatant of Aspergillus oryzae RIB40 culture. We cloned the genes encoding AME1 and AME2 based on the genomic information of A. oryzae, constructed their expression systems in A. oryzae, and obtained the recombinant enzymes (rAME1 and rAME2). rAME1 did not act on the KGM polymer but only on the KGM oligomer, releasing approximately 60% of the acetic acid in the substrate. However, rAME2 was active against both KGM substrates, releasing approximately 80% and 100% of acetic acid from the polymer and oligomer, respectively. Both enzymes were active against xylan and exhibited a trace activity on ethyl ferulate. The acetyl group position specificities of both enzymes were analyzed via heteronuclear single quantum correlation NMR using oligosaccharides of glucomannan prepared from Aloe vera (AGM), which has a higher acetyl group content than KGM. rAME1 acted specifically on single-substituted acetyl groups and not on double-substituted ones. In contrast, rAME2 appeared to act on all the acetyl groups in AGM. Treatment of 3% KGM with rAME2 followed by heating to 90 °C resulted in gel formation under weakly acidic conditions. This is the first study to induce gelation of KGM under these conditions. A comparison of the breaking and brittleness properties of gels formed by alkaline and enzymatic treatments revealed similar texture of the two gels. Furthermore, scanning electron microscopy of the surface structure of both gels revealed that both formed a fine mesh structure. Our findings on enzymatic gelation of KGM should lead to the development of new applications in food manufacturing industry.

Visible Light Degradation of a Monoclonal Antibody in a High-Concentration Formulation: Characterization of a Tryptophan-Derived Chromophoric Photo-product by Comparison to Photo-degradation of N-Acetyl-l-tryptophan Amide.

We investigated the discoloration of a highly concentrated monoclonal antibody (mAbZ) in sodium acetate (NaAc) and histidine/lysine (His/Lys) buffer after exposure to visible light. The color change of the mAbZ formulation was significantly more intense in NaAc buffer and developed a characteristic absorbance with a λmax of ca. 450 nm. We characterized this photo-chemically generated chromophore by comparison with visible light photo-degradation of a concentrated solution of a model compound for protein Trp residues, N-acetyl-l-tryptophan amide (NATA). The photo-degradation of NATA generated a chromophoric product with a λmax of ca. 450 nm and UV-vis spectroscopic properties identical to those of the product generated from mAbZ. This product was isolated and analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and 1H, 13C, and 1H-13C heteronuclear single-quantum correlation NMR spectroscopy. MS/MS analysis reveals a product characterized by the loss of 33 Da from NATA, referred to as NATA-33. Together, the NMR data suggest that this product may be N-(2,4-dihydrocyclopenta[b]indol-2-yl)acetamide (structure P3a) or a tautomer (P3b-d).

Establishing Drug Discovery and Identification of Hit Series for the Anti-apoptotic Proteins, Bcl-2 and Mcl-1.

We describe our work to establish structure- and fragment-based drug discovery to identify small molecules that inhibit the anti-apoptotic activity of the proteins Mcl-1 and Bcl-2. This identified hit series of compounds, some of which were subsequently optimized to clinical candidates in trials for treating various cancers. Many protein constructs were designed to identify protein with suitable properties for different biophysical assays and structural methods. Fragment screening using ligand-observed NMR experiments identified several series of compounds for each protein. The series were assessed for their potential for subsequent optimization using 1H and 15N heteronuclear single-quantum correlation NMR, surface plasmon resonance, and isothermal titration calorimetry measurements to characterize and validate binding. Crystal structures could not be determined for the early hits, so NMR methods were developed to provide models of compound binding to guide compound optimization. For Mcl-1, a benzodioxane/benzoxazine series was optimized to a Kd of 40 μM before a thienopyrimidine hit series was identified which subsequently led to the lead series from which the clinical candidate S 64315 (MIK 665) was identified. For Bcl-2, the fragment-derived series were difficult to progress, and a compound derived from a published tetrahydroquinone compound was taken forward as the hit from which the clinical candidate (S 55746) was obtained. For both the proteins, the work to establish a portfolio of assays gave confidence for identification of compounds suitable for optimization.

Membrane separation and characterisation of lignin and its derived products obtained by a mild ethanol organosolv treatment of rice straw

Abstract An organosolv process using ethanol-water was optimized in order to recover high quality lignin from rice-straw previously pre-treated by autohydrolysis at 210 °C. The results showed a selective and appreciable removal of lignin under very mild conditions and the highest delignification yield occurred at 30 °C. The lignin extracts were characterised using capillary zone electrophoresis (CZE), size exclusion chromatography (SEC), Fourier transform infrared spectroscopy (FT-IR) and 31P-NMR, and two-dimensional heteronuclear single quantum correlation NMR spectroscopy (2D-HSQC NMR), which enabled the identification of low molecular weight lignins with a syringyl/guaiacyl ratio of about 0.74 containing phenolic compounds with potential bioactive properties. In order to separate the target compounds, membrane technology has been used and an enriched extract containing value-added phenolic compounds such as tricin, vanillin, ferulic acid and p-coumaric acid was obtained. High membrane efficiency (around 80%) was obtained for target compounds.

Thermal stability of low and high Mw fractions of bio-oil derived from lignin conversion in subcritical water

The thermal stability of bio-oil influences its application in industry and is, therefore, a very important factor that must be taken into consideration. In this study, the stability of low and high molecular weight (Mw) fractions of bio-oil obtained from the hydrothermal liquefaction (HTL) of lignin in subcritical water was studied at an elevated temperature (80 °C) for a period of 1 h, 1 day and 1 week. The changes in molecular weight (gel permeation chromatography (GPC)) and chemical composition (gas chromatography–mass spectrometry (GC–MS) and 2D heteronuclear single quantum correlation (HSQC) NMR (18.8 T, DMSO-d6)) of low and high Mw fractions of the HTL bio-oil (i.e. light oil (LO) and heavy oil (HO)) were evaluated before and after ageing. It was found that only a slight formation of high Mw insoluble structures was obtained during ageing at elevated temperature for 1 week: 0.5% for the LO and 3.1% for the HO. These higher Mw moieties might be formed from different polymerisation/condensation reactions of the reactive compounds (i.e. anisoles, guaiacols, phenols, methylene (–CH2–) groups in phenolic dimers and xanthene). The high Mw insolubles in both the LO and the HO were analysed for structural composition using 2D HSQC NMR to obtain a better understanding of the changes in the composition of bio-oil fractions during the accelerated ageing process. In addition, a chemical shift database in DMSO-d6 was analysed for a subset of phenolic model compounds to simplify the interpretation of the 2D HSQC NMR spectra.

Surface Properties of Squalene/Meibum Films and NMR Confirmation of Squalene in Tears.

Squalene (SQ) possesses a wide range of pharmacological activities (antioxidant, drug carrier, detoxifier, hydrating, emollient) that can be of benefit to the ocular surface. It can come in contact with human meibum (hMGS; the most abundant component of the tear film lipid layer) as an endogenous tear lipid or from exogenous sources as eyelid sebum or pharmaceuticals. The aims of this study were to determine (i) if SQ is in tear lipids and (ii) its influence on the surface properties of hMGS films. Heteronuclear single quantum correlation NMR confirmed 7 mol % SQ in Schirmer’s strips extracts. The properties of SQ/hMGS pseudo-binary films at the air/water interface were studied with Langmuir surface balance, stress-relaxation dilatational rheology and Brewster angle microscopy. SQ does not possess surfactant properties. When mixed with hMGS squalene (i) localized over the layers’ thinner regions and (ii) did not affect the film pressure at high compression. Therefore, tear SQ is unlikely to instigate dry eye, and SQ can be used as a safe and “inert” ingredient in formulations to protect against dry eye. The layering of SQ over the thinner film regions in addition to its pharmacological properties could contribute to the protection of the ocular surface.

Selective hydrolysis of hen egg white lysozyme at Asp-X peptide bonds promoted by oxomolybdate

The activity of oxomolybdate(VI) towards hen egg white lysozyme (HEWL) was examined under physiological and slightly acidic pH conditions. Purely hydrolytic cleavage of HEWL in the presence of 10 to 100mM of oxomolybdate(VI) after incubation at pH5.0 and 60°C for 2 to 7days was observed in SDS-PAGE experiments. Four cleavage sites, which all occurred at Asp-X sequences and included the Asp18-Asn19, Asp48-Gly49, Asp52-Trp53 and Asp101-Gly102 peptide bonds, were identified with Edman degradation. The molecular interaction between [MoO4]2− and HEWL was studied by circular dichroism (CD) and 1H-15N heteronuclear single quantum correlation (HSQC) NMR spectroscopy. CD spectroscopy revealed a significant decrease in the α-helical content of HEWL upon addition of oxomolybdate, while 1H-15N HSQC NMR spectroscopy identified the residues which were most affected upon interaction with [MoO4]2−. 95Mo NMR measurements, performed on oxomolybdate solutions containing HEWL, identified the monomeric [MoO4]2− form as active species in the hydrolytic reaction. The hydrolysis of the Asp-Gly model peptide in the presence of oxomolybdate(VI) was studied by 1H NMR, further supporting a hydrolytic mechanism where polarisation of the carbonyl is followed by internal nucleophilic attack on the Asp residue.

Solution structure of lysine‐free (K0) ubiquitin

Lysine-free ubiquitin (K0-Ub) is commonly used to study the ubiquitin-signaling pathway, where it is assumed to have the same structure and function as wild-type ubiquitin (wt-Ub). However, the K0-Ub (15) N heteronuclear single quantum correlation NMR spectrum differs significantly from wt-Ub and the melting temperature is depressed by 19°C, raising the question of the structural integrity and equivalence to wt-Ub. The three-dimensional structure of K0-Ub was determined by solution NMR, using chemical shift and residual dipolar coupling data. K0-Ub adopts the same backbone structure as wt-Ub, and all significant chemical shifts can be related to interactions impacted by the K to R mutations.

Rapid Heteronuclear Single Quantum Correlation NMR Spectra at Natural Abundance

A novel NMR experiment, the so-called ASAP-HSQC, is introduced that allows the detection of heteronuclear one-bond correlations in less than 30 s on small molecules at natural abundance without compromises in sweep width, resolution or spectral quality. Equally, the experiment allows a significant increase in digital resolution or a moderate senstitivity enhancement in the same overall experiment time compared to a conventional HSQC. The gain is a consequence of keeping all unused proton magnetization along z during acquisition, so that the previously reported ASAP and ALSOFAST approaches can be transferred from HMQC to HSQC-type experiments. Next to basic and broadband pulse sequences, a characterization of the sequence with respect to minimum measurement time, sensitivity gain, and advantages in resolution compared to state-of-the-art experiments is given.

The Arabidopsis B3 Domain Protein VERNALIZATION1 (VRN1) Is Involved in Processes Essential for Development, with Structural and Mutational Studies Revealing Its DNA-binding Surface

The B3 DNA-binding domain is a plant-specific domain found throughout the plant kingdom from the alga Chlamydomonas to grasses and flowering plants. Over 100 B3 domain-containing proteins are found in the model plant Arabidopsis thaliana, and one of these is critical for accelerating flowering in response to prolonged cold treatment, an epigenetic process called vernalization. Despite the specific phenotype of genetic vrn1 mutants, the VERNALIZATION1 (VRN1) protein localizes throughout the nucleus and shows sequence-nonspecific binding in vitro. In this work, we used a dominant repressor tag that overcomes genetic redundancy to show that VRN1 is involved in processes beyond vernalization that are essential for Arabidopsis development. To understand its sequence-nonspecific binding, we crystallized VRN1(208-341) and solved its crystal structure to 1.6 Å resolution using selenium/single-wavelength anomalous diffraction methods. The crystallized construct comprises the second VRN1 B3 domain and a preceding region conserved among VRN1 orthologs but absent in other B3 domains. We established the DNA-binding face using NMR and then mutated positively charged residues on this surface with a series of 16 Ala and Glu substitutions, ensuring that the protein fold was not disturbed using heteronuclear single quantum correlation NMR spectra. The triple mutant R249E/R289E/R296E was almost completely incapable of DNA binding in vitro. Thus, we have revealed that although VRN1 is sequence-nonspecific in DNA binding, it has a defined DNA-binding surface.

On the road to improve the bioremediation and electricity-harvesting skills of Geobacter sulfurreducens: functional and structural characterization of multihaem cytochromes

Extracellular electron transfer is one of the physiological hallmarks of Geobacter sulfurreducens, allowing these bacteria to reduce toxic and/or radioactive metals and grow on electrode surfaces. Aiming to functionally optimize the respiratory electron-transfer chains, such properties can be explored through genetically engineered strains. Geobacter species comprise a large number of different multihaem c-type cytochromes involved in the extracellular electron-transfer pathways. The functional characterization of multihaem proteins is particularly complex because of the coexistence of several microstates in solution, connecting the fully reduced and oxidized states. NMR spectroscopy has been used to monitor the stepwise oxidation of each individual haem and thus to obtain information on each microstate. For the structural study of these proteins, a cost-effective isotopic labelling of the protein polypeptide chains was combined with the comparative analysis of 1H-13C HSQC (heteronuclear single-quantum correlation) NMR spectra obtained for labelled and unlabelled samples. These new methodological approaches allowed us to study G. sulfurreducens haem proteins functionally and structurally, revealing functional mechanisms and key residues involved in their electron-transfer capabilities. Such advances can now be applied to the design of engineered haem proteins to improve the bioremediation and electricity-harvesting skills of G. sulfurreducens.

Single-scan 2D NMR correlations by multiple coherence transfers

A new scheme for the acquisition of heteronuclear 2D correlations in NMR spectroscopy within a single scan, is proposed and demonstrated. The principles of this new scheme resemble those of Mansfield’s “k-space walk” proposal, in the sense that they rely on repetitively transferring spin coherences back-and-forth between the two spin systems to be correlated. It is shown that if properly executed, these transfers enable the equivalent of a continuous sampling of the time-domain space supporting a 2D heteronuclear single-quantum correlation NMR spectrum. Details on how to execute the resulting “time-domain walk” experiments are given, and examples comparing it against conventional and other single-scan 2D acquisition alternatives are shown. Advantages, opportunities, and main drawbacks of this new ultrafast approach to 2D NMR, are briefly discussed.

Proton-detected heteronuclear single quantum correlation NMR spectroscopy in rigid solids with ultra-fast MAS

In this article, we show the potential for utilizing proton-detected heteronuclear single quantum correlation (HSQC) NMR in rigid solids under ultra-fast magic angle spinning (MAS) conditions. The indirect detection of carbon-13 from coupled neighboring hydrogen nuclei provides a sensitivity enhancement of 3- to 4-fold in crystalline amino acids over direct-detected versions. Furthermore, the sensitivity enhancement is shown to be significantly larger for disordered solids that display inhomogeneously broadened carbon-13 spectra. Latrodectus hesperus (Black Widow) dragline silk is given as an example where the sample is mass-limited and the sensitivity enhancement for the proton-detected experiment is 8- to 13-fold. The ultra-fast MAS proton-detected HSQC solid-state NMR technique has the added advantage that no proton homonuclear decoupling is applied during the experiment. Further, well-resolved, indirectly observed carbon-13 spectra can be obtained in some cases without heteronuclear proton decoupling.

Automatic compatibility tests of HSQC NMR spectra with proposed structures of chemical compounds

A recently introduced similarity measure is extended here for comparing two-dimensional spectra. Its applicability is demonstrated with heteronuclear single-quantum correlation (HSQC) NMR spectra. For testing the compatibility of a spectrum with the proposed chemical structure, first, the spectrum is predicted on the basis of that structure and then, the proposed comparison algorithm is applied. In this context, the topics of optimization are peak picking, signal intensity measures, and optimizing the parameters of the two-dimensional comparison method. The performance is analyzed with a test set of 289 structures of organic compounds and their HSQC and 1H NMR spectra. The results obtained with HSQC spectra are better than those achieved using the previously described one-dimensional similarity test with 1H NMR spectra alone.

Biochemical, Functional, and Pharmacological Characterization of AT-56, an Orally Active and Selective Inhibitor of Lipocalin-type Prostaglandin D Synthase

We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3–250 μm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH2 (Km = 14 μm) with a Ki value of 75 μm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD2 by L-PGDS-expressing human TE-671 cells after stimulation with Ca2+ ionophore (5 μm A23187) with an IC50 value of about 3 μm without affecting their production of PGE2 and PGF2α but had no effect on the PGD2 production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD2 production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE2 and PGF2α and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.

Probing the Interaction between the Coiled Coil Leucine Zipper of cGMP-dependent Protein Kinase Iα and the C Terminus of the Myosin Binding Subunit of the Myosin Light Chain Phosphatase

Nitric oxide and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase I (PKG-Ialpha)-mediated activation of myosin phosphatase (MLCP). Mechanistically it has been proposed that protein-protein interactions between the N-terminal leucine zipper (LZ) domain of PKG-Ialpha ((PKG-Ialpha(1-59)) and the LZ and/or coiled coil (CC) domain of the myosin binding subunit (MBS) of MLCP are localized in the C terminus of MBS. Although recent studies have supported these interactions, the critical amino acids responsible for these interactions have not been identified. Here we present structural and biophysical data identifying that the LZ domain of PKG-Ialpha(1-59) interacts with a well defined 42-residue CC motif (MBS(CT42)) within the C terminus of MBS. Using glutathione S-transferase pulldown experiments, chemical cross-linking, size exclusion chromatography, circular dichroism, and isothermal titration calorimetry we identified a weak dimer-dimer interaction between PKG-Ialpha(1-59) and this C-terminal CC domain of MBS. The K(d) of this non-covalent complex is 178.0+/-1.5 microm. Furthermore our (1)H-(15)N heteronuclear single quantum correlation NMR data illustrate that this interaction is mediated by several PKG-Ialpha residues that are on the a, d, e, and g hydrophobic and electrostatic interface of the C-terminal heptad layers 2, 4, and 5 of PKG-Ialpha. Taken together these data support a role for the LZ domain of PKG-Ialpha and the CC domain of MBS in this requisite contractile complex.

Comparative Biophysical Characterization of p53 with the Pro-apoptotic BAK and the Anti-apoptotic BCL-xL

The p53 transcription-independent apoptosis in mitochondria, mediated by its interaction with the pro-apoptotic and the anti-apoptotic members of the Bcl2 family of proteins, has been described in vivo, especially in radiosensitive tissues. We have characterized the interaction of p53 with both the pro-apoptotic Bak and the anti-apoptotic Bcl-x(L) proteins, comparing their affinity and their interaction surfaces, using biophysical techniques such as fluorescence anisotropy, analytical ultracentrifugation, and NMR. We have shown that both proteins interact with only the p53 core domain and not with its N- and C-terminal regions. Further, p53 has a higher affinity for Bcl-x(L) than for Bak, which is consistent with the previously described sequential binding of Bcl-x(L) and Bak by p53. Interestingly, although the interaction with both proteins is electrostatic in character, they have different binding sites. Using NMR spectroscopy, we have determined that Bcl-x(L) interacts with the DNA binding site of p53, but Bak does not interact with this site. A new potential interaction surface for Bak is proposed.