The Stejskal-Tanner (ST) equation enables quantitative analysis of molecular diffusion in diffusion-ordered spectroscopy (DOSY), yet its derivation becomes increasingly complex when extended to modern pulse sequences especially under experimental conditions such as convection. Here, we present a systematic theoretical framework to explain and derive the ST equation. Central to this work is a Mathematica-based implementation of the derivation, which decomposes the calculation into three generalizable steps applicable to pulsed-gradient spin echo (POSE), pulsed-gradient stimulated echo (POSTE), bipolar pulse pair stimulated echo (BPP-STE), and convection-compensated BPP-double STE sequences. Fully executable Mathematica code is provided for each sequence, enabling rapid and reproducible derivation of the corresponding gradient-dependent attenuation expressions. This work aims to provide both conceptual clarity and computational codes, supporting accurate determination of diffusion coefficients across a wide range of DOSY experiments.
Licorice (Glycyrrhiza spp.) is an important medicinal plant, and a new hybrid cultivar, Wongam (G. korshinskyi), has recently been developed in Korea through interspecific hybridization between G. uralensis and G. glabra. In this study, 1H NMR based metabolomic approach was employed to compare the primary metabolite profiles of Wongam and G. uralensis. A total of 33 primary metabolites, including sugars, organic acids, and amino acids, were identified and quantified. Multivariate statistical analyses revealed clear metabolic discrimination between the two cultivars. In particular, sucrose, malate, and trigonelline were more abundant in G. uralensis, whereas lactate, acetate, and several amino acid-related metabolites were present at higher levels in Wongam, indicating their potential as discriminative markers for licorice differentiation. These findings provide fundamental insight into the primary metabolic characteristics of licorice and establish a basis for future studies on quality evaluation and breeding improvement.
The M9 minimal medium is widely used for producing isotope-labeled proteins in Escherichia coli (E. coli). However, the original M9 formulation does not fully support optimal E. coli growth. To address this limitation, several modified versions featuring increased potassium concentrations, enhanced buffering capacity, and other improvements have been developed to promote robust growth and high-yield protein production. In this protocol, we aimed to identify the best conditions that support E. coli survival and adaptation by comparing the performance of modified minimal medium variants with the original M9 medium, and proposed the optimized formulation of a minimal medium (M-opt) suitable for efficient isotope-labeled protein production.
Solid-state nuclear magnetic resonance (NMR) spectroscopy is widely used to investigate the structure and dynamics of a broad range of materials. In the case of paramagnetic materials, interactions with unpaired electrons lead to markedly broadened NMR frequency ranges, increased linewidths, and shortened longitudinal relaxation times (T-1). These features necessitate experimental approaches and spectral interpretation strategies that differ from those used for diamagnetic systems. In this paper, we summarize key spectroscopic characteristics of paramagnetic materials in solid-state NMR and present several representative examples illustrating these phenomena.
beta-Ga2O3 single crystals co-doped with Fe3+ and Mn2+ impurity ions were grown using the floating zone method. By employing an X-band electron magnetic resonance (EMR) spectrometer, electron magnetic resonance spectra of Fe3+ impurity ions in the host crystals were recorded at room temperature. The rotation patterns of Fe3+ resonance lines in the crystallographic planes clearly show that the real local site symmetry of Fe3+ ion within the crystal is monoclinic. It turns out that the Fe3+ impurity ions substitute for Ga3+ ions (Ga3+I sites) in the oxygen octahedron in fl-Ga2O3 single crystal doped with Fe and Mn ions. The substitutional sites of Fe3+ ions are discussed and compared with Mn2+, Cr3+, and Gd3+ impurity ions in the host fl-Ga2O3 crystals.
Cereblon (CRBN) is a substrate receptor of the CRL4 E3 ubiquitin ligase complex and a key component in the mechanism of action of immunomodulatory drugs (IMiDs) such as pomalidomide. In this study, we investigated the molecular interaction between pomalidomide and the C-terminal thalidomide-binding domain of CRBN (CdCRBN) using solution NMR spectroscopy, isothermal titration calorimetry (ITC), and fluorescence-based competition assays. Two glycine residues, G354 and G383, located near the ligandbinding site, were employed as markers to monitor binding-induced chemical shift perturbations. The 1H, 15N-HSQC titration revealed concentration-dependent chemical shift changes, indicating fast exchange kinetics. The dissociation constant (Kd) calculated by NMR was 55 +/- 1.8 mu M, while ITC analysis showed a stronger binding affinity (Kd = 12.5 mu M), suggesting an enthalpy-driven interaction. A fluorescence resonance energy transfer (FRET)-based competition assay using MANT-uracil further supported the interaction, yielding a Ki of 2.1 mu M. These results confirm that pomalidomide binds to the canonical thalidomide-binding pocket of CdCRBN, providing a structural and thermodynamic basis for its function as a molecular glue.
The key intermediate 5 of the AB-CHMINACA metabolite M2 was synthesized through a sequence of alkylation, hydrolysis, and amide coupling reactions, affording a moderate yield. In particular, the compound 2 and its regioisomer 3 were distingushed by Nuclear Overhouse Effect (NOE) experiments. In the NOE analysis, compound 3 show no NOE correlation between the methylene (CH2) and the aromatic proton, whereas compound 2 exhibited a pronounced NOE effect. The unambiguous identification of compound 2 was crucial for the synthesis of the AB-CHMINACA metabolite M2. The resulting sy nthetic metabolite will serve as a reference standard compound for the detection of illicit drugs.
Galdieria sulphuraria is a thermoacidophilic red alga that thrives in extreme environments and encodes numerous hypothetical proteins with uncharacterized functions. In this study, we describe the recombinant production, purification, and isotopic labeling of a truncated construct (residues 1-130) of the hypothetical protein Gasu_38600 for NMR-based structural analysis. The construct was expressed in Escherichia coli using a maltose-binding protein fusion system and purified via immobilized metal affinity chromatography followed by TEV protease cleavage. Uniform 15N and [13C,15N] isotopic labeling was achieved using minimal media supplemented with 15NH4Cl and 13C-glucose. High-quality 1H-15N HSQC spectra indicated that the protein adopts a folded conformation in solution, supporting its suitability for further structural investigation. These results lay the groundwork for future studies to elucidate the function of this conserved hypothetical protein from an extremophilic organism.
Fluoxetine, a common antidepressant and an environmental contaminant, was investigated for its effects on zebrafish larvae and adults using NMR-based metabolomics. Adult zebrafish were exposed to fluoxetine concentrations of 70 and 700 mu g/L for 72 hours, while larvae were exposed to 7 and 70 mu g/L from 1 to 72 hours post-fertilization. Our analysis revealed alterations in 77 metabolites in adults and 58 in larvae, with both groups showing changes in citrate, serine, and glutathione levels, and decrease in succinate and fumarate, highlighting some conserved metabolic responses to fluoxetine. Pathway analysis indicated significant effects on the TCA cycle, as well as glycine, serine, and threonine metabolism across both developmental stages. Notably, larvae exhibited distinct shifts with increased glutamine and decrease in glutamate and glycine, suggesting heightened sensitivity during early development. In adults, specific changes included increases in ATP, N,N-dimethylglycine, and alanine, with decreases in phenylalanine, tyrosine, betaine, NADP+ and 3-hydroxyisovalerate, pointing to possible metabolic disruptions. These alterations affected key pathways, notably the glycerolipid pathway in larvae and tyrosine and beta-alanine metabolism in adults, indicating stage-specific susceptibility to fluoxetine. Furthermore, the application of HR-MAS NMR has proven instrumental in elucidating the complex metabolic responses to fluoxetine exposure, underscoring its utility in biotoxicity assessments.
Nuclear magnetic resonance (NMR) spectroscopy is a versatile tool for investigating various physicochemical properties of biomolecules. Among diverse applications of biomolecular NMR spectroscopy, F-19-based methodology has recently attracted much attention from researchers in protein science owing to its advantageous applicability to proteins of high molecular weights or heterogeneous structural states. In this mini-review, the recent application example of F-19 NMR to transthyretin, a representative amyloidogenic protein, thus being an important yet challenging target for structural investigation, is summarized along with some perspectives for its future directions.
Toxins produced by marine toxigenic algae have garnered growing attention due to their detrimental impacts on marine ecosystem, aquaculture, and human health. Among these, diarrhetic shellfish poisoning (DSP) toxins, such as okadaic acid (OA), are of particular concern. In this study, we report the successful isolation and structural elucidation of three new derivatives of OA from the marine dinoflagellate Prorocentrum lima. . These newly identified compounds, OA-2Me-C7, OA-2-Me-C8, and OA-1-Me-C8, were characterized through a comprehensive series of NMR experiments, combined with structural comparisons to the well-known OA. The identification of these derivatives contributes to the expanding knowledge of DSP toxin diversity and provides new insights into the structural variations of these harmful algal toxins.
Caveolin3, mainly expressed in muscle tissue types, is a structural scaffolding protein of caveolae which are microdomains of plasma membrane. To elucidate the relationship between structure and function, several studies on the structure of caveolins using NMR have been reported. Because the ionic strength can affect the electrostatic-driven association of proteins with ligand and protein structure, the effect of salt in the structural studies has to be considered. In this work, we observed that the chemical shifts of Cav3 in the LPPG detergent change depending on salt concentration. The R2 values also show salt concentration-dependent changes. Specifically, in the N-terminal region where conformational changes and various interactions occur, the R2 values decrease. Interestingly, the R2 values of residues expected to be located in the LPPG detergent are also influenced by the salt concentration. This work suggests that the concentration of NaCl can affect interpretation of NMR data from membrane proteins.
Epstein-Barr virus (EBV) Latent Membrane Protein 2A (LMP2A) is a transmembrane protein essential for viral latency and immune evasion, structured with distinct functional domains. The N-terminal domain modulates B-cell receptor signaling, while the transmembrane and C-terminal regions help anchor the protein and facilitate additional interactions within host cells. This study characterizes the LMP2A C-terminal domain (CTD) structure under various conditions using CD spectroscopy and NMR. The 29-residue C-tail sequence contains key residues that impact stability. CD analysis shows that, while disordered at neutral pH and in water, helicity is slightly enhanced under acidic conditions and membrane-mimicking environments, with DTT influencing stability through oxidation state effects. NMR structural analysis in DPC micelles reveals a well-converged helical structure extending from residues R471 to E486, closely matching AlphaFold predictions. The data highlight the CTD's structural plasticity and suggest roles for cysteines and hydrophobic clusters in stabilizing the peptide in membrane-like settings.
Based on projected Liouville equation, we theoretically investigated the magnetic field dependence of the magneto-optical cyclotron transition of the quasi two-dimensional Landau splitting system in GaAs and CdS. Through the numerical analysis of the current work, we found the increasing properties of line-widths of GaAs and CdS with magnetic field. We also found that line-widths of GaAs < CdS in the magnetic field region of B > 20.06 T and presented the reasonable resonating pictures of the line-widths.
The peptide hormone glucagon has served as both a biopharmaceutical agent for clinical use and a model peptide forming amyloid fibrils. As we recently established a recombinant production of glucagon, its backbone NMR assignments at neutral pHs were conducted in the present study. However, as the NMR spectra showed severe line broadening and poor dispersion at pHs 7.5 and 7.0, complete backbone NMR assignments could be obtained at pH 6.0. Interestingly, CSI and TALOS predictions using the assigned chemical shift values indicated different probabilities of secondary structure. These results suggest that glucagon would adopt an unusual or dynamically fluctuating conformation in solution. Therefore, detailed conformation and molecular dynamics of glucagon would be worthy of investigation, for which the present results provide an experimental basis.
Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), enhances serotonin levels in synapses to alleviate depressive symptoms. This study investigates the effects of fluoxetine on the aquatic organism Daphnia magna (D. magna), known for its environmental sensitivity. D. magna was exposed to 100 mu g/L of fluoxetine for 72 hours, and metabolomic changes were examined using high-resolution magic angle spinning (HR-MAS) NMR. A total of twentyeight metabolites were identified. Compared to the control group, fluoxetine exposure resulted in decreased levels of phenylalanine and increased levels of sn-glycero- 3-phosphocholine, inosine, proline, carnosine, and tryptophan.
The binding feature of bipyrrole-strapped calix[4]pyrrole 1 for the fluoride anion was examined by a combination of H-1 and F-19 NMR spectroscopy in DMSO-d(6) . Receptor 1 was found to bind the fluoride anion with high affinity via two step-process. Fluoride binding to receptor 1 was proved to facilitate deuterium exchange of the pyrrolic protons in the nominally aprotic deuterated solvent of DMSO-d(6).
This article presents various features related to a strange, yet extremely important group of proteins called intrinsically disordered proteins (IDPs) which used to be and are still by some called generically "unstructured" proteins. Huge abundance (similar to 30% of the entire human proteome) of IDPs has brought a critical paradigm shift in protein science as well as in structural biology during the last three decades. In this article a brief introduction on these unorthodox proteins will be followed by an explanation for "the long-and-winding" journey to reach the consensus name of IDP. The term IDP was adopted with difficulty only in 2012 after 20 years since the first discovery reports on "unstructured" proteins had appeared. Next, some comments will be made on the hurdles encountered in the early days to have the concept of "unstructured" proteins or the manuscripts dealing with them accepted in the protein science field. Thirdly, the saga by the KRIBB NMR team to establish the pre-structured motifs (PreSMos) that are the transient secondary structures observed in the target unbound state of IDPs as the target-binding active sites of IDPs will be delineated in depth along with the relevant publications. Finally, the issues that need to be explored further in the IDP field such as AlphaFold will be discussed.
IscU, the iron -sulfur (Fe -S) cluster scaffold protein, is an essential protein for biogenesis of Fe -S clusters. Previous studies showed that IscU manifests a metamorphic structural feature; at least two structural states, namely the structured state (S -state) and the disordered state (D -state), interconverting in a physiological condition, was observed. Moreover, subsequent studies demonstrated that the metamorphic flexibility of IscU is important for its Fe -S cluster assembly activity as well as for an efficient interaction with various partner proteins. Although solution nuclear magnetic resonance (NMR) spectroscopy has been a useful tool to investigate this protein, the detailed molecular mechanism that sustains the structural heterogeneity of IscU is still unclear. To tackle this issue, we applied a high-pressure NMR (HP-NMR) technique to the IscU variant, IscU(I8K), which shows an increased population of the S -state. We found that the equilibrium between the S- and Dstate was significantly perturbed by pressure application, and the specific regions of IscU exhibited more sensitivity to pressure than the other regions. Our results provide novel insights to appreciate the dynamic behaviors of IscU and the related versatile functionality.
In a study utilizing NMR spectroscopy and chemometrics, propolis samples from seven diverse geographic regions across Turkey were analyzed. To identify the optimal method for studying both the antimicrobial properties and compositional variations of propolis from different regions, we investigated metabolite extraction using three solvents: water only, ethanol only, and sequential water-ethanol extraction for residual components. Notably, water-soluble components exhibited significant variation among the samples, which is particularly interesting considering the potability of propolis in water-based solutions. Furthermore, the Mugla sample displayed a distinct water-soluble profile, likely due to its unique coastal location on the Aegean Sea. This specific climate may influence the propolis' chemical composition, resulting in a different mixture of components. Interestingly, the Mugla sample contained pharmaceutically active compounds like cinnamate, ferulate, and verapamil. This research establishes valuable foundation for further exploration of propolis' antimicrobial potential.
