Structure Characterization with NMR Molecular Networking

Nuclear Magnetic Resonance (NMR) is among the most widely used techniques for structure determination, yet automated workflows remain underdeveloped compared to mass spectrometry. In this work, we introduce NMR molecular networking and apply it to Heteronuclear Single Quantum Coherence (HSQC) spectra, a key 2D-NMR experiment for structure elucidation. We adapt core principles of MS² networking such as transitivity across multiple spectra, dereplication, and annotation propagation to NMR-driven workflows. First, we develop a modified Hungarian distance metric for HSQC peak matching. Benchmarks show that using this metric, traditional spectral lookup with this score recovers ~70-80% of available structural similarity, but efficiency does not improve when increasing the size of the spectral library. Second, we establish NMR molecular networking using HSQC spectra to propagate annotations and dereplicate compounds. Case studies of experimental natural product spectra demonstrate that annotation transitivity within networks accelerates and improves identification of unknowns. Third, we introduce algorithmic molecular networking, which integrates graph topology metrics to correct inefficient rankings and reduce false positives. Together, these approaches define the first generalizable framework for NMR molecular networking, enabling scalable, high-throughput annotation for natural product discovery and drug development.

SNARE protein complexes are key mediators of exocytosis by juxtaposing opposing membranes, leading to membrane fusion. SNAREs generally consist of one or two core domains that can form a four-helix bundle with other SNARE core domains. Some SNAREs, such as syntaxin target-SNAREs and longin vesicular-SNAREs, have independent, folded N-terminal domains that can interact with their respective SNARE core domains and thereby affect the kinetics of SNARE complex formation. This autoinhibition mechanism is believed to regulate the role of the longin VAMP7/TI-VAMP in neuronal morphogenesis. Here we use nuclear magnetic resonance spectroscopy to study the longin-SNARE core domain interaction for VAMP7. Using complete backbone resonance assignments, chemical shift perturbations analysis, and hydrogen/deuterium exchange experiments, we conclusively show that VAMP7 adopts a preferentially closed conformation in solution. Taken together, the closed conformation of longins is conserved, in contrast to the syntaxin family of SNAREs for which mixtures of open and closed states have been observed. This may indicate different regulatory mechanisms for SNARE complexes containing syntaxins and longins, respectively.

Misfolding of the natively α-helical prion protein into a β-sheet rich isoform is related to various human diseases such as Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. In humans, the disease phenotype is modified by a methionine/valine polymorphism at codon 129 of the prion protein gene. Using a combination of hydrogen/deuterium exchange coupled to NMR spectroscopy, hydroxyl radical probing detected by mass spectrometry, and site-directed mutagenesis, we demonstrate that stop mutants of the human prion protein have a conserved amyloid core. The 129 residue is deeply buried in the amyloid core structure, and its mutation strongly impacts aggregation. Taken together the data support a critical role of the polymorphic residue 129 of the human prion protein in aggregation and disease.

A simple, rapid, in vitro cell-free protein expression system, Expressway NMR, is introduced and used to express the small ubiquitin-related modifier protein SUMO-1. This 12 kDa molecule is challenging for NMR as it has limited solubility and requires relatively high salt (200 mM) for stability in solution. Starting with the gene, the cell-free system, and milligram amounts of nitrogen-15 isotopically enriched amino acids, sufficient protein is produced in 4 h to obtain a high-resolution 2D HSQC spectrum of the protein in 40 min. This time would be closer to 10 min with the aid of a higher sensitivity salt-tolerant cryogenic NMR probe. With all protein purification steps included, and aggressive data processing using the filter diagonalization method (FDM), it is but 6 h from gene to heteronuclear single quantum coherence (HSQC). As the cell-free system is nearly background-free, it is also possible to work with the crude reaction mixture, in which case only a total of 5 h is required. Sample stability over time, whether crude extract or purified, was notable, with no significant change in the 15N-1H HSQC spectrum over 6 months at 4 degrees C (300 muM, pH 6.1, capped NMR tube). The combination of a turnkey, high-yield, protease-free in vitro protein expression system, an optimized sensitivity-enhanced HSQC pulse sequence, and FDM processing makes this scheme an attractive first step to rapidly assess the suitability of proteins for complete solution structure determination.

Decision letter: Dynamic Na+/H+ exchanger 1 (NHE1) – calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation

A small 63-residue membrane protein, p7, has essential roles in the infectivity of the hepatitis C virus in humans. This hydrophobic membrane protein forms homo-oligomeric ion channels in bilayers, which can be blocked by known channel-blocking compounds. To perform structural studies of p7 by nuclear magnetic resonance (NMR) spectroscopy, it is necessary to produce milligram quantities of isotopically labeled protein; as is the case for most membrane-associated proteins, this is challenging. We describe the successful expression of full-length p7 and two truncated constructs in Escherichia coli using a fusion partner that directs the overexpressed protein to inclusion bodies. Following isolation of the fusion proteins by affinity chromatography, they were chemically cleaved with cyanogen bromide. The p7-polypeptides were purified by size-exclusion chromatography. Solution NMR two-dimensional heteronuclear single quantum coherence spectra of uniformly (15) N-labeled p7-polypeptides in 1,2-dihexyl-1-sn-glycero-3-phosphocholine isotropic micelles are fully resolved, with a single resonance for each amide site. The solid-state NMR spectra of the same polypeptides in magnetically aligned 14-O-PC/6-O-PC bicelles demonstrate their reconstitution into planar phospholipid bilayers.

Comprehensive analysis of isotopic labeling patterns of metabolites in proteinogenic amino acids and starch for plant systems lay in the powerful tool of 2-Dimensional [(1)H, (13)C] Nuclear Magnetic Resonance (2D NMR) spectroscopy. From (13)C-labeling experiments, 2D NMR provides information on the labeling of particular carbon positions, which contributes to the quantification of positional isotope isomers (isotopomer). 2D Heteronuclear Single Quantum Correlation (HSQC) NMR distinguishes particularly between the labeling patterns of adjacent carbon atoms, and leads to a characteristic enrichment of each carbon atom of amino acids and glucosyl and mannosyl units present in hydrolysates of glycosylated protein. Furthermore, this technique can quantitatively classify differences in glucosyl units of starch hydrolysate and of protein hydrolysate of plant biomass. Therefore, the 2D HSQC NMR method uses proteinogenic amino acids and starch to provide an understanding of carbon distribution of compartmentalization in the plant system. NMR has the advantage of minimal sample handle without separate individual compounds prior to analysis, for example multiple isotopomers can be detected, and their distribution extracted quantitatively from a single 2D HSQC NMR spectrum. The peak structure obtained from the HSQC experiment show multiplet patterns, which are directly related to isotopomer balancing. These abundances can be translated to maximum information on the metabolic flux analysis. Detailed methods for the extractions of protein, oil, soluble sugars, and starch, hydrolysis of proteinogenic amino acid and starch, and NMR preparation using soybean embryos cultured in vitro as a model plant systems are reported in this text. In addition, this chapter includes procedures to obtain the relative intensity of 16 amino acids and glucosyl units from protein hydrolysate and the glucosyl units of starch hydrolysate of soybean embryos in 2D HSQC NMR spectra.

Cyclic AMP receptor protein (CRP) is a homodimeric protein, which is activated by cAMP binding to function as a transcriptional regulator of many genes in prokaryotes. Until now, the actual number of cAMP molecules that can be bound by CRP in solution has been ambiguous. In this work, we performed a nuclear magnetic resonance study on CRP to investigate the stoichiometry of cyclic nucleotide binding to CRP. A series of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of the protein in the absence and in the presence of cAMP or cGMP were analyzed. The addition of cAMP to CRP induced a biphasic spectral change up to 4 equivalents, whereas the cGMP addition made a monophasic change up to 2 equivalents. Altogether, the results not only established for the first time that CRP possesses two cyclic AMP-binding sites in each monomer, even in a solution without DNA, but also suggest that the syn-cAMP binding sites of the CRP dimer can be formed by an allosteric conformational change of the protein upon the binding of two anti-cAMPs at the N-terminal domain. In addition, a residue-specific inspection of the spectral changes provides some new structural information about the cAMP-induced allosteric activation of CRP.

A sulfonated aromatic block copolymer (SABC), consisting of hydrophobic and hydrophilic blocks, was analyzed by heteronuclear single-quantum correlation (HSQC), heteronuclear multiple-bond correlation (HMBC) and HSQC total correlation spectroscopy (HSQC–TOCSY). Because of its complicated chemical structure with five different phenylene rings, 12 types of 1H signals and 24 types of 13C signals were observed in a narrow chemical shift range (7.0–8.0 p.p.m. for 1H and 118–162 p.p.m. for 13C). To improve the 1H signal separation, the temperature conditions for the 1H nuclear magnetic resonance (NMR) experiments were optimized. Moreover, 1H and 13C NMR signal assignments for the hydrophobic blocks were performed using HSQC and HMBC, with reference to the assignments of a model oligomer. For the hydrophilic blocks, furthermore, HSQC–TOCSY techniques were applied. As a result of these studies, complete 1H and 13C NMR signal assignments were made for the SABC. The ion-exchange capacity (IEC) and the copolymerization composition were calculated using the 1H NMR assignments for the SABC, and the IEC value obtained in this way was consistent with that obtained via titration. A sulfonated aromatic block copolymer (SABC), consisting of hydrophobic and hydrophilic blocks, was analyzed by 2D NMR techniques of HSQC, HMBC and HSQC–TOCSY. Because of its complicated chemical structure with five different phenylene rings, 12 types of 1H signals and 24 types of 13C signals were observed in a narrow chemical shift range (7.0–8.0 p.p.m. for 1H and 118–162 p.p.m. for 13C). As a result of these studies, complete 1H and 13C NMR signal assignment of SABC was performed.

Identification of the synthetic cannabinoid N-(2-phenyl-propan-2-yl)-1-(4-cyanobutyl)-1H-indazole-3-carboxamide (CUMYL-4CN-BINACA) in a herbal mixture product

<p>Marine algae are an important and historically rich source of new marine-based natural products. This thesis describes the screening of 40 Indonesian macroalgal samples using liquid chromatography-mass spectrometry (LC-MS) based molecular networking, and the subsequent nuclear magnetic resonance (NMR)-guided isolation and structural elucidation of a 6-deoxy-6-aminoglycoglyrecolipid (60). Molecular networking was performed using LC-MS/MS data through the online Global Natural Product Social Molecular Networking (GNPS) platform directly from crude extracts. NMR spectroscopy-guided screening was also employed targeting unique peaks detected by 1H NMR to validate any hits from GNPS. Out of 40 macroalgae specimens collected from West Timor waters, six samples were prioritised by the molecular networking screening. Proton NMR revealed three specimens with significantly interesting peaks but only one specimen, Laurencia snackeyi was purified further, which yielded compound 60.</p>

Accurate identification of lipids in biological samples is a key step in lipidomics studies. Multidimensional nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool for this purpose as it provides comprehensive structural information on lipid composition at atomic resolution. However, the interpretation of NMR spectra of complex lipid mixtures is currently hampered by limited spectral resolution and the absence of a customized lipid NMR database along with user-friendly spectral analysis tools. We introduce a new two-dimensional (2D) NMR metabolite database "COLMAR Lipids" that was specifically curated for hydrophobic metabolites presently containing 501 compounds with accurate experimental 2D 13C-1H heteronuclear single quantum coherence (HSQC) chemical shift data measured in CDCl3. A new module in the public COLMAR suite of NMR web servers was developed for the (semi)automated analysis of complex lipidomics mixtures (http://spin.ccic.osu.edu/index.php/colmarm/index2). To obtain 2D HSQC spectra with the necessary high spectral resolution along both 13C and 1H dimensions, nonuniform sampling in combination with pure shift spectroscopy was applied allowing the extraction of an abundance of unique cross-peaks belonging to hydrophobic compounds in complex lipidomics mixtures. As shown here, this information is critical for the unambiguous identification of underlying lipid molecules by means of the new COLMAR Lipids web server, also in combination with mass spectrometry, as is demonstrated for Caco-2 cell and lung tissue cell extracts.

This study aims to analyse the phytochemistry of two medicinal plants locally used by herbalists Pavetta crassipes leaves and Calotropis procera bark from Malawi. The specific objectives of the study are: To extract Pavetta crassipes leaves and Calotropis procera bark using 80% methanol and 100% methanol respectively. To isolate compounds using open column and medium pressure liquid chromatography. To elucidate the structures of isolated pure compounds using nuclear magnetic resonance spectral techniques. The research design was an experimental method. The dried Pavetta crassipes leaves (2900g) and Calotropis procera bark (4500g) was extracted with 3L 80% methanol and 100% MeOH respectively, at room temperature after 72 hours. Using various column chromatographical techniques each of the extracts was isolated to get the pure compounds. The isolated pure compounds were elucidated and characterized using Proton Nuclear magnetic Resonance Spectrum (1H NMR), Carbon Nuclear Magnetic Resonance (13C NMR), Heteronuclear Multiple Bond Coherence (HMBC), Heteronuclear Single Quantum Coherence (HSQC), Distortionless Enhancement via Polarization Transfer (DEPT), Nuclear Overhauser Effect Spectroscopy (NOESY) and Correlation Spectroscopy (COSY). The elucidation of pure compounds from Pavetta classipes yielded flavonoid Quercetin-3-O-rutinoside (1), Chlorogenic acid (2) and Methyl chlorogenate (3) and Calotropis procera yielded taraxasteryl acetate (4) and calactin (5).

1H High-Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) is a reliable technology used for detecting metabolites in solid tissues. Fast response time enables guiding surgeons in real time, for detecting tumor cells that are left over in the excision cavity. However, severe overlap of spectral resonances in 1D signal often render distinguishing metabolites impossible. In that case, Heteronuclear Single Quantum Coherence Spectroscopy (HSQC) NMR is applied which can distinguish metabolites by generating 2D spectra ( 1H- 13C). Unfortunately, this analysis requires much longer time and prohibits real time analysis. Thus, obtaining 2D spectrum fast has major implications in medicine. In this study, we show that using multiple multivariate regression and statistical total correlation spectroscopy, we can learn the relation between the 1H and 13C dimensions. Learning is possible with small sample sizes and without the need for performing the HSQC analysis, we can predict the 13C dimension by just performing 1H HRMAS NMR experiment. We show on a rat model of central nervous system tissues (80 samples, 5 tissues) that our methods achieve 0.971 and 0.957 mean R2 values, respectively. Our tests on 15 human brain tumor samples show that we can predict 104 groups of 39 metabolites with 97 percent accuracy. Finally, we show that we can predict the presence of a drug resistant tumor biomarker (creatine) despite obstructed signal in 1H dimension. In practice, this information can provide valuable feedback to the surgeon to further resect the cavity to avoid potential recurrence.

The use of a ditopic Gd(III) paramagnetic probe for investigating α-bungarotoxin surface accessibility