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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]

SHIP2 is a multi-domain inositol 5-phosphatase binding to a variety of phosphotyrosine (pY)-containing proteins through its SH2 domain, so as to regulate various cell signaling pathways by modulating the phosphatidylinositol level in the plasma membrane. Unfavorably, Helicobacter pylori can hijack SHIP2 through the CagA protein to induce gastric cell carcinogenesis. To date, the interaction between SHIP2 and CagA was not analyzed from a structural point of view. Here, the binding of SHIP2-SH2 with Tyr-phosphorylated peptides from four EPIYA motifs (A/B/C/D) in CagA was studied using NMR spectroscopy. The results showed that EPIYA-C and -D bind to a similar interface of SHIP2-SH2, including a pY-binding pocket and a hydrophobic pocket, to achieve high affinity, while EPIYA-A and -B bind to a smaller interface of SHIP2-SH2 with weak affinity. By summarizing the interface and affinity of SHIP2-SH2 for CagA EPIYA-A/B/C/D, c-MET and FcgR2B ITIM, it was proposed that, potentially, SHIP2-SH2 has a selective preference for L > I > V for the aliphatic residues at the pY+3 position in its ligand. This study reveals the rule of the ligand sequence bound by SHIP2-SH2 and the mechanism by which CagA protein hijacks SHIP2, which will help design a peptide inhibitor against SHIP2-SH2. [ABSTRACT FROM AUTHOR]

α-Synuclein (α-syn) is the main protein component of Lewy bodies, the major pathological hallmarks of Parkinson's disease (PD). C-terminally truncated α-syn is found in the brain of PD patients, reduces cell viability and tends to form fibrils. Nevertheless, little is known about the mechanisms underlying the role of C-terminal truncation on the cytotoxicity and aggregation of α-syn. Here, we use nuclear magnetic resonance spectroscopy to show that the truncation alters α-syn conformation, resulting in an attractive interaction of the N-terminus with membranes and molecular chaperone, protein disulfide isomerase (PDI). The truncated protein is more toxic to mitochondria than full-length protein and diminishes the effect of PDI on α-syn fibrillation. Our findings reveal a modulatory role for the C-terminus in the cytotoxicity and aggregation of α-syn by interfering with the N-terminus binding to membranes and chaperone, and provide a molecular basis for the pathological role of C-terminal truncation in PD pathogenesis. C-terminal truncation of a-syn results in a more extended and exposed conformation, providing further insight into the pathological role of this truncation event in the progression of Parkinson's disease. [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]

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]

Abstract: Most proteins function in cells where protein concentrations can reach 400g/l. However, most quantitative studies of protein properties are performed in idealized, dilute conditions. Recently developed in-cell NMR techniques can provide protein structure and other biophysical properties inside living cells at atomic resolution. Here we review how protein dynamics, including global and internal motions have been characterized by in-cell NMR, and then discuss the remaining challenges and future directions. [Copyright &y& Elsevier]

Conformational change of cytochrome c (cyt c) caused by interaction with cardiolipin (CL) is an important step during apoptosis, but the underlying mechanism is controversial. To comprehensively clarify the structural transformations of cyt c upon interaction with CL and avoid the unpredictable alias that might come from protein labeling or mutations, the conformation of purified yeast iso–1 cyt c with natural isotopic abundance in different contents of CL was measured by using NMR spectroscopy, in which the trimethylated group of the protein was used as a natural probe. The data demonstrate that cyt c has two partially unfolded conformations when interacted with CL: one with Fe–His33 coordination and the other with a penta–coordination heme. The Fe–His33 coordination conformation can be converted into a penta–coordination heme conformation in high content of CL. The structure of cyt c becomes partially unfolded with more exposed heme upon interaction with CL, suggesting that cyt c prefers a high peroxidase activity state in the mitochondria, which, in turn, makes CL easy to be oxidized, and causes the release of cyt c into the cytoplasm as a trigger in apoptosis. [ABSTRACT FROM AUTHOR]

Clostridium perfringens autolysin (CpAcp) is a peptidoglycan hydrolase associated with cell separation, division, and growth. It consists of a signal peptide, ten SH3b domains, and a catalytic domain. The structure and function mechanisms of the ten SH3bs related to cell wall peptidoglycan binding remain unclear. Here, the structures of CpAcp SH3bs were studied through NMR spectroscopy and structural simulation. The NMR structure of SH3b6 was determined at first, which adopts a typical β-barrel fold and has three potential ligand-binding pockets. The largest pocket containing eight conserved residues was suggested to bind with peptide ligand in a novel model. The structures of the other nine SH3bs were subsequently predicted to have a fold similar to SH3b6. Their ligand pockets are largely similar to those of SH3b6, although with varied size and morphology, except that SH3b1/2 display a third pocket markedly different from those in other SH3bs. Thus, it was supposed that SH3b3-10 possess similar ligand-binding ability, while SH3b1/2 have a different specificity and additional binding site for ligand. As an entirety, ten SH3bs confer a capacity for alternatively binding to various peptidoglycan sites in the cell wall. This study presents an initial insight into the structure and potential function of CpAcp SH3bs. [ABSTRACT FROM AUTHOR]

Abstract: α-Synuclein is involved in Parkinson's disease and its interaction with cell membrane is crucial to its pathological and physiological functions. Membrane properties, such as curvature and lipid composition, have been shown to affect the interactions by various techniques, but ion effects on α-synuclein membrane interactions remain elusive. Ca2+ dynamic fluctuation in neurons plays important roles in the onset of Parkinson's disease and its influx is considered as one of the reasons to cause cell death. Using solution Nuclear Magnetic Resonance (NMR) spectroscopy, here we show that Ca2+ can modulate α-synuclein membrane interactions through competitive binding to anionic lipids, resulting in dissociation of α-synuclein from membranes. These results suggest a negative modulatory effect of Ca2+ on membrane mediated normal function of α-synuclein, which may provide a clue, to their dysfunction in neurodegenerative disease. [Copyright &y& Elsevier]