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Metabolism is a fundamental process that underlies human health and diseases. Nuclear magnetic resonance (NMR) techniques offer a powerful approach to identify metabolic processes and track the flux of metabolites at the molecular level in living systems. An in vitro study through in-cell NMR tracks metabolites in real time and investigates protein structures and dynamics in a state close to their most natural environment. This technique characterizes metabolites and proteins involved in metabolic pathways in prokaryotic and eukaryotic cells. In vivo magnetic resonance spectroscopy (MRS) enables whole-organism metabolic monitoring by visualizing the spatial distribution of metabolites and targeted proteins. One limitation of these NMR techniques is the sensitivity, for which a possible improved approach is through isotopic enrichment or hyperpolarization methods, including dynamic nuclear polarization (DNP) and parahydrogen-induced polarization (PHIP). DNP involves the transfer of high polarization from electronic spins of radicals to surrounding nuclear spins for signal enhancements, allowing the detection of low-abundance metabolites and real-time monitoring of metabolic activities. PHIP enables the transfer of nuclear spin polarization from parahydrogen to other nuclei for signal enhancements, particularly in proton NMR, and has been applied in studies of enzymatic reactions and cell signaling. This review provides an overview of in-cell NMR, in vivo MRS, and hyperpolarization techniques, highlighting their applications in metabolic studies and discussing challenges and future perspectives. [ABSTRACT FROM AUTHOR]

Few reports are found working on the features and functions of the human telomere G-triplex (ht-G3) though the telomere G-quadruplex has been intensely studied and widely implemented to develop various biosensors. We herein report that ht-G3 lights up Thioflavin T (ThT) and establish a sensitive biosensing platform for RNA detection by introducing a target recycling strategy. An optimal condition was selected out for ht-G3 to promote ThT to generate a strong fluorescence. Accordingly, an ht-G3-based molecular beacon was successfully designed against the corresponding RNA sequence of the SARS-CoV-2 N-gene. The sensitivity for the non-amplified RNA target achieves 0.01 nM, improved 100 times over the conventional ThT–based method. We believe this ht-G3/ThT–based label-free strategy could be widely applied for RNA detection. [ABSTRACT FROM AUTHOR]

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) plays an essential role in regulating phosphatidylinositol level in human cell, and is recruited to many phosphotyrosine (pY)-dependent signal transduction pathways by the SH2 domain. In immunity signaling, immunoreceptor FcγRIIB binds to SHIP2-SH2 via its Y292-phosphorylated immunoreceptor tyrosine-based inhibitory motif (ITIM) and transmits inhibitory signal, which regulates B cell and neuronal cell activity and is associated with immune diseases and Alzheimer's disease. To date, the interaction between SHIP2 and FcγRIIB has not been analyzed from a structural point of view. Here, the binding of SHIP2-SH2 with Y292-phosphorylated FcγRIIB-ITIM was analyzed using NMR spectroscopy. The results demonstrated that SHIP2-SH2 mainly utilizes two regions including a pY-binding pocket and a specificity pocket formed by βD, βE, and EF-loop, to bind with FcγRIIB-ITIM in high affinity. In addition to the two regions, the BG-loop of SHIP2-SH2 functions as an auxiliary interface enhancing affinity. By comparing the binding of SHIP2-SH2 with ligands from FcγRIIB and c-MET, a hepatocyte growth factor receptor associated with tumorigenesis, significant differences in interface and affinity were found, suggesting that SHIP2-SH2 applies diverse patterns for binding to different ligand proteins. Moreover, S49, S51, and R70 of SHIP2 were identified to mediate the binding of both FcγRIIB and c-MET, while R28 and Q107 were found to only participate in the binding of c-MET and FcγRIIB respectively. Taken together, this study reveals the diverse mechanisms of SHIP2-SH2 for recognizing different ligands, and provides important clues for selectively manipulating various signaling pathways and specific drug design. [ABSTRACT FROM AUTHOR]

To eliminate the effects of complex background signals and to enhance the accuracy of the diffusion coefficient measurement, derivative NMR spectroscopy with negligible loss of the spectral quality is introduced based on the customized Savitzky-Golay method and used to construct diffusion-ordered NMR spectroscopy (DOSY). The criterion of the method was established by simulations. The application of this method on mouse urine and serum showed that the accuracy and precision of diffusion coefficient measurements in a complex background were improved to enhance the identification of molecules. [ABSTRACT FROM AUTHOR]

Ionic liquids have been extensively used as environmentally friendly solvents for enzymatic reactions and other biological systems. Understanding the mechanism of how ionic liquids affect protein stability is crucial for the biological reaction processes and protein storage using ionic liquids as solvents. Although effects of ionic liquids on protein stability have been studied, equilibrium thermodynamics of protein stability in ionic liquids has not been quantitatively measured. Herein, we utilized 19F NMR to measure the equilibrium thermodynamics of protein stability in ionic liquid [C4-mim]Br. Our results show that proteins are significantly destabilized in [C4-mim]Br ionic liquids. Our results suggest that 19F NMR provides a simple and effective way to study the thermodynamics of protein stability in ionic liquids. 19F NMR will be applicable to facilitate the protein–protein interaction study in ionic liquids. [ABSTRACT FROM AUTHOR]

Protein diffusion in living cells might differ significantly from that measured in vitro. Little is known about the effect of globular protein size on rotational diffusion in cells because each protein has distinct surface properties, which result in different interactions with cellular components. To overcome this problem, the B1 domain of protein G (GB1) and several concatemers of the protein were labeled with 5-fluorotryptophan and studied by F NMR in Escherichia coli cells, Xenopus laevis oocytes, and in aqueous solutions crowded with glycerol, or Ficoll70™ and lysozyme. Relaxation data show that the size dependence of protein rotation in cells is due to weak interactions of the target protein with cellular components, but the effect of these interactions decreases as protein size increases. The results provide valuable information for interpreting protein diffusion data acquired in living cells. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography are the two main methods for protein three-dimensional structure determination at atomic resolution. According to the protein structures deposited in the Protein Data Bank, X-ray crystallography has become the dominant method for structure determination, particularly for large proteins and complexes. However, with the developments of isotope labeling, increase of magnetic field strength, common use of a cryogenic probe, and ingenious pulse sequence design, the applications of NMR spectroscopy have expanded in biological research, especially in characterizing protein dynamics, sparsely populated transient structures, weak protein interactions, and proteins in living cells at atomic resolution, which is difficult if not impossible by other biophysical methods. Although great advances have been made in protein NMR spectroscopy, its applications in protein therapeutics, which represents the fastest growing segment of the pharmaceutical industry, are still limited. Here we review the recent advances in the use of NMR spectroscopy in studies of large proteins or complexes, posttranslation modifications, weak interactions, and aggregation, and in-cell NMR spectroscopy. The potential applications of NMR spectroscopy in protein therapeutic assays are discussed. [ABSTRACT FROM AUTHOR]