Conventional structural biology has largely relied on purified proteins studied under artificial in vitro conditions, which cannot fully recapitulate the crowded, heterogeneous, and dynamic intracellular environment. In-cell NMR spectroscopy overcomes this limitation by enabling atomic-resolution observation of protein structures, dynamics, and interactions directly inside living cells. This review summarizes recent advances in in-cell NMR methodologies, focusing on strategies for preparing isotope-labeled target proteins in different cellular contexts. We discuss in situ expression approaches in prokaryotic, insect, and mammalian cells, as well as exogenous delivery methods for mammalian in-cell NMR, including electroporation, cell-penetrating peptides, and reversible membrane permeabilization using streptolysin O. We further highlight representative applications in the characterization of intrinsically disordered proteins, protein maturation, and intracellular interactions. Future integration with bioreactor-based sample systems, microfluidics, cryoelectron tomography, and AI-assisted structural modeling is expected to expand the scope and impact of in-cell NMR. By preserving the native cellular context of proteins, in-cell NMR offers a powerful platform for understanding biomolecular function and pharmacology inside living cells.
F-19 nuclear magnetic resonance (NMR) spectroscopy has been acknowledged as a powerful method for investigating the structure, dynamics, and related functional features of challenging protein systems. Because fluorine is absent in native biological systems, F-19 NMR provides highly sensitive, background-free spectra that alleviate the molecular weight limitations and spectral crowding, common yet challenging issues to conventional biomolecular NMR. Following our F-19 NMR review series, in this mini-review, we examine and compare the principles, advantages, limitations, and key applications of widely adopted protein F-19 labeling strategies: biosynthetic incorporation of fluorinated amino acids, cysteine-targeted modification, genetic code expansion, transglutaminase-mediated enzymatic labeling of glutamines, and lysine-targeted modification. This comprehensive comparison establishes a practical guide for selecting optimal F-19 labeling strategies for challenging pharmacological targets and macromolecular complexes.
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.
