RESUMEN

Skeletal muscle is closely linked to energy metabolism, but it is inevitably deprived of energy. Cellular differentiation is an essential and energy-demanding process in skeletal muscle development. Much attention has been paid to identifying beneficial factors that promote skeletal muscle satellite cell differentiation and further understanding the underlying regulatory mechanisms. As a critical metabolic substrate or regulator, α-ketoglutarate (AKG) has been recognized as a potential nutritional supplement or therapeutic target for skeletal muscle. We have previously found beneficial effects of AKG supplementation on the proliferation of C2C12 myoblasts cultured under both normal and energy-deficient conditions and have further elucidated the underlying metabolic mechanisms. However, it remains unclear what role AKG plays in myotube formation in different energy states. In the present study, we investigated the effects of AKG supplementation on the differentiation of C2C12 myoblasts cultured in normal medium (Nor myotubes) and low glucose medium (Low myotubes) and performed NMR-based metabonomic profiling to address AKG-induced metabolic changes in both Nor and Low myotubes. Significantly, AKG supplementation promoted myotube formation and induced metabolic remodeling in myotubes under normal medium and low glucose medium, including improved energy metabolism and enhanced antioxidant capacity. Specifically, AKG mainly altered amino acid metabolism and antioxidant metabolism and upregulated glycine levels and antioxidase expression. Our results are typical for the mechanistic understanding of the effects of AKG supplementation on myotube formation in the two energy states. This study may be beneficial for further exploring the applications of AKG supplementation in sports, exercise, and therapy.

Asunto(s)

RESUMEN

Phosphatase and tensin homolog is a lipid phosphatase that serves as the major negative regulator of the PI3K/AKT pathway. It catalyzes the 3'-specific dephosphorylation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to generate PIP2. PTEN's lipid phosphatase function depends on several domains, including an N-terminal segment spanning the first 24 amino acids, which results in a catalytically impaired enzyme when mutated. Furthermore, PTEN is regulated by a cluster of phosphorylation sites located on its C-terminal tail at Ser380, Thr382, Thr383, and Ser385, which drives its conformation from an open to a closed autoinhibited but stable state. Herein, we discuss the protein chemical strategies we used to reveal the structure and mechanism of how PTEN's terminal regions govern its function.

Asunto(s)

RESUMEN

The water-soluble blue-green pigment marennine, produced and partly excreted by the diatom Haslea ostrearia, and known for a long time for its role in the greening of oysters, was isolated from the culture medium, purified, and analyzed by Nuclear Magnetic Resonance (NMR) in order to gain insight into its chemical structure. The spectra show mainly carbohydrates of a complex composition, apparently highly branched, and with a mass in the order of 10 kDa. There are, in addition, some signals of aliphatic and, much weaker, aromatic groups that present aglycons. The latter might be responsible for the color. These carbohydrates are always associated with the blue-green color and cannot be separated from it by most treatments; they are interpreted as constituting the frame of the pigment. NMR after hydrolysis identifies the most abundant monosaccharides in marennine as galactose, xylose, mannose, rhamnose, and fucose.

Asunto(s)

RESUMEN

NMR is a valuable experimental tool in the structural biologist's toolkit to elucidate the structures, functions, and motions of biomolecules. The progress of machine learning, particularly in structural biology, reveals the critical importance of large, diverse, and reliable datasets in developing new methods and understanding in structural biology and science more broadly. Biomolecular NMR research groups produce large amounts of data, and there is renewed interest in organizing these data to train new, sophisticated machine learning architectures and to improve biomolecular NMR analysis pipelines. The foundational data type in NMR is the free-induction decay (FID). There are opportunities to build sophisticated machine learning methods to tackle long-standing problems in NMR data processing, resonance assignment, dynamics analysis, and structure determination using NMR FIDs. Our goal in this study is to provide a lightweight, broadly available tool for archiving FID data as it is generated at the spectrometer, and grow a new resource of FID data and associated metadata. This study presents a relational schema for storing and organizing the metadata items that describe an NMR sample and FID data, which we call Spectral Database (SpecDB). SpecDB is implemented in SQLite and includes a Python software library providing a command-line application to create, organize, query, backup, share, and maintain the database. This set of software tools and database schema allow users to store, organize, share, and learn from NMR time domain data. SpecDB is freely available under an open source license at https://github.rpi.edu/RPIBioinformatics/SpecDB.

Asunto(s)

RESUMEN

The 3J coupling values are commonly used in biomolecular NMR to extract structural information. Here we present a novel intra HNCA IP/AP E.COSY pulse sequence that allows to measure 3J HNHa coupling constants by a simple and rapid two-dimensional 1H-15N correlation experiment where the 15N frequency is encoded at the same time as the 1J HaCa coupling evolution. The advantage with respect to the conventional 3D HNCA E.COSY pulse sequence is the dimensionality reduction to a simple 2D experiment, which decreases acquisition time and facilitates data analysis. The performance of this new experiment is demonstrated with an ubiquitin sample at 500 MHz.

Asunto(s)

RESUMEN

Magnetic resonance spectroscopy (MRS), as a noninvasive method for molecular structure determination and metabolite detection, has grown into a significant tool in clinical applications. However, the relatively low signal-to-noise ratio (SNR) limits its further development. Although the multichannel coil and repeated sampling are commonly used to alleviate this problem, there is still potential room for promotion. One possible improvement way is combining these two acquisition methods so that the complementary of them can be well utilized. In this paper, a novel coil-combination method, average smoothing singular value decomposition, is proposed to further improve the SNR by introducing repeatedly sampled signals into multichannel coil combination. Specifically, the sensitivity matrix of each sampling was pretreated by whitened singular value decomposition (WSVD), then the smoothing was performed along the repeated samplings' dimension. By comparing with three existing popular methods, Brown, WSVD, and generalized least squares, the proposed method showed better performance in one phantom and 20 in vivo spectra.

RESUMEN

Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.

Asunto(s)

RESUMEN

α-Ketoglutarate (AKG) is attracting much attention from researchers owing to its beneficial effects on anti-aging and cancer suppression, and, more recently, in nutritional supplements. Given that glucose is the main source of energy to maintain normal physiological functions of skeletal muscle, the effects of AKG supplementation for improving muscle performance are closely related to the glucose level in skeletal muscle. The differences of AKG-induced effects in skeletal muscle between two states of normal energy and energy deficiency are unclear. Furthermore, AKG-induced metabolic changes in skeletal muscles in different energy states also remain elusive. Here, we assessed the effects of AKG supplementation on mouse C2C12 myoblast cells cultured both in normal medium (Nor cells) and in low-glucose medium (Low cells), which were used to mimic two states of normal energy and energy deficiency, respectively. We further performed NMR-based metabolomic analysis to address AKG-induced metabolic changes in Nor and Low cells. AKG supplementation significantly promoted the proliferation and differentiation of cells in the two energy states through glutamine metabolism, oxidative stress, and energy metabolism. Under normal culture conditions, AKG up-regulated the intracellular glutamine level, changed the cellular energy status, and maintained the antioxidant capacity of cells. Under low-glucose culture condition, AKG served as a metabolic substrate to reduce the glutamine-dependence of cells, remarkably enhanced the antioxidant capacity of cells and significantly elevated the intracellular ATP level, thereby ensuring the normal growth and metabolism of cells in the state of energy deficiency. Our results provide a mechanistic understanding of the effects of AKG supplements on myoblasts in both normal energy and energy deficiency states. This work may be beneficial to the exploitation of AKG applications in clinical treatments and nutritional supplementations.

Asunto(s)

RESUMEN

Ultra-fast magic-angle spinning (UFMAS) at a MAS rate (ωR/2π) of 60 kHz or higher has dramatically improved the resolution and sensitivity of solid-state NMR (SSNMR). However, limited polarization transfer efficiency using cross-polarization (CP) between 1H and rare spins such as 13C still restricts the sensitivity and multi-dimensional applications of SSNMR using UFMAS. We propose a novel approach, which we call decoherence-optimized tilted-angle CP (DOTA CP), to improve CP efficiency with prolonged lifetime of 1H coherence in the spin-locked condition and efficient band-selective polarization transfer by incorporating off-resonance irradiation to 1H spins. 13C CP-MAS at ωR/2π of 70-90 kHz suggested that DOTA CP notably outperformed traditional adiabatic CP, a de-facto-standard CP scheme over the past decade, in sensitivity for the aliphatic-region spectra of 13C-labeled GB1 protein and N-formyl-Met-Leu-Phe samples by up to 1.4- and 1.2-fold, respectively. 1H-detected 2D 1H/13C SSNMR for the GB1 sample indicated the effectiveness of this approach in various multidimensional applications.

Asunto(s)

RESUMEN

Combining dynamic nuclear polarization with proton detection significantly enhances the sensitivity of magic-angle spinning NMR spectroscopy. Herein, the feasibility of proton-detected experiments with slow (10 kHz) magic angle spinning was demonstrated. The improvement in sensitivity permits the acquisition of indirectly detected 14 N NMR spectra allowing biomolecular structures to be characterized without recourse to isotope labelling. This provides a new tool for the structural characterization of environmental and medical samples, in which isotope labelling is frequently intractable.

RESUMEN

Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. Here we describe a photo-catalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements.

Asunto(s)

RESUMEN

The newly identified CUBAN (Cullin binding domain associating with NEDD8) domain recognizes both ubiquitin and the ubiquitin-like NEDD8. Despite the high similarity between the two molecules, CUBAN shows a clear preference for NEDD8, free and conjugated to cullins. We previously characterized the domain structure, both alone and in complex with NEDD8. The results here reported are addressed to investigate the determinants that drive the selective binding of CUBAN towards NEDD8 and ubiquitin. The 15N HSQC NMR perturbation pattern of the labeled CUBAN domain, when combined with either NEDD8 or ubiquitin, shows a clear involvement of hydrophobic residues that characterize the early stages of these interactions. After a slow conformational selection step, hydrophobic and then neutral and polar interactions take place, which drive the correct orientation of the CUBAN domain, leading to differences in the recognition scheme of NEDD8 and ubiquitin. As a result, a cascade of induced fit steps seems to determine the structural preference shown for NEDD8 and therefore the basis of the selectivity of the CUBAN domain. Finally, molecular dynamics analysis was performed to determine by fluctuations the internal flexibility of the CUBAN/NEDD8 complex. We consider that our results, based on a structural investigation mainly focused on the early stages of the recognition, provide a fruitful opportunity to report the different behavior of the same protein with two highly similar binding partners.

Asunto(s)

RESUMEN

Escherichia coli expression protocols for selective labeling of methyl groups in proteins have been essential in expanding the size range of targets that can be studied by biomolecular NMR. Based on the initial work achieving selective labeling of isoleucine, leucine, and valine residues, additional methods were developed over the past years which enabled the individual and/or simultaneous combinatorial labeling of all methyl containing amino acids. Together with the introduction of new methyl-optimized NMR experiments, this now allows the detailed characterization of protein-ligand interactions as well as mechanistic and dynamic processes of protein-protein complexes up to 1MDa in size. In this chapter, we provide a general introduction to selective labeling of proteins using E. coli-based expression systems, describe the considerations taken into account prior to the selective labeling of a protein, and include the protocols used to produce such proteins. An overview of applications using selectively labeled proteins with an emphasis on examples relevant to the drug discovery process is then presented.

Asunto(s)

RESUMEN

The HypF protein is involved in the maturation and regulation of hydrogenases. The N-terminal domain of HypF (HypF-N) has served as a key model system to study the pathways of protein amyloid formation and the nature of the toxicity of pre-fibrilar protein oligomers. This domain can aggregate into two forms of oligomers having significantly different toxic effects when added to neuronal cultures. Here, NMR assignments of HypF-N backbone resonances are presented in its native state and under the conditions favouring the formation of toxic and non-toxic oligomers. The analyses of chemical shifts provide insights into the protein conformational state and the possible pathways leading to the formation of different types of oligomers.

Asunto(s)

RESUMEN

Dynamic nuclear polarization (DNP) is a powerful way to overcome the sensitivity limitation of magic-angle-spinning (MAS) NMR experiments. However, the resolution of the DNP NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures and in the presence of paramagnetic radicals. High-quality DNP-enhanced NMR spectra of the Acinetobacter phage 205 (AP205) nucleocapsid can be obtained by combining high magnetic field (800 MHz) and fast MAS (40 kHz). These conditions yield enhanced resolution and long coherence lifetimes allowing the acquisition of resolved 2D correlation spectra and of previously unfeasible scalar-based experiments. This enables the assignment of aromatic resonances of the AP205 coat protein and its packaged RNA, as well as the detection of long-range contacts, which are not observed at room temperature, opening new possibilities for structure determination.

RESUMEN

Protein-Observed Fluorine NMR (PrOF NMR) spectroscopy is an emerging technique for screening and characterizing small-molecule-protein interactions. The choice of which amino acid to label for PrOF NMR can be critical for analysis. Here we report the first use of a protein containing two different fluoroaromatic amino acids for NMR studies. Using the KIX domain of the CBP/p300 as a model system, we examine ligand binding of several small-molecule compounds elaborated from our previous fragment screen and identify a new ligand binding site distinct from those used by native transcription factors. This site was further supported by computational modeling (FTMap and Schrödinger) and 1 H,15 N HSQC/HMQC NMR spectroscopy. Metabolic labeling with multiple fluorinated amino acids provides useful probes for further studying ligand binding and has led to new insight for allosterically regulating transcription-factor protein interactions with small-molecule ligands.

Asunto(s)

RESUMEN

Solid-state NMR (ssNMR) can provide structural information at the most detailed level and, at the same time, is applicable in highly heterogeneous and complex molecular environments. In the last few years, ssNMR has made significant progress in uncovering structure and dynamics of proteins in their native cellular environments [1-4]. Additionally, ssNMR has proven to be useful in studying large biomolecular complexes as well as membrane proteins at the atomic level [5]. In such studies, innovative labeling schemes have become a powerful approach to tackle spectral crowding. In fact, selecting the appropriate isotope-labeling schemes and a careful choice of the ssNMR experiments to be conducted are critical for applications of ssNMR in complex biomolecular systems. Previously, we have introduced a software tool called FANDAS (Fast Analysis of multidimensional NMR DAta Sets) that supports such investigations from the early stages of sample preparation to the final data analysis [6]. Here, we present a new version of FANDAS, called FANDAS 2.0, with improved user interface and extended labeling scheme options allowing the user to rapidly predict and analyze ssNMR data sets for a given protein-based application. It provides flexible options for advanced users to customize the program for tailored applications. In addition, the list of ssNMR experiments that can be predicted now includes proton (1H) detected pulse sequences. FANDAS 2.0, written in Python, is freely available through a user-friendly web interface at http://milou.science.uu.nl/services/FANDAS .

Asunto(s)

RESUMEN

The chemical shifts measured in solution-state and solid-state nuclear magnetic resonance (NMR) are powerful probes of the structure and dynamics of protein molecules. The exploitation of chemical shifts requires methods to correlate these data with the protein structures and sequences. We present here an approach to calculate accurate chemical shifts in both ordered and disordered proteins using exclusively the information contained in their sequences. Our sequence-based approach, protein sequences and chemical shift correlations (PROSECCO), achieves the accuracy of the most advanced structure-based methods in the characterization of chemical shifts of folded proteins and improves the state of the art in the study of disordered proteins. Our analyses revealed fundamental insights on the structural information carried by NMR chemical shifts of structured and unstructured protein states.

Asunto(s)

RESUMEN

The editors of this special volume suggested this topic, presumably because of the perspective lent by our combined >90-year association with biomolecular NMR. What follows is our personal experience with the evolution of the field, which we hope will illustrate the trajectory of change over the years. As for the future, one can confidently predict that it will involve unexpected advances. Our narrative is colored by our experience in using the NMR Facility for Biomedical Studies at Carnegie-Mellon University (Pittsburgh) and in developing similar facilities at Purdue (1977-1984) and the University of Wisconsin-Madison (1984-). We have enjoyed developing NMR technology and making it available to collaborators and users of these facilities. Our group's association with the Biological Magnetic Resonance data Bank (BMRB) and with the Worldwide Protein Data Bank (wwPDB) has also been rewarding. Of course, many groups contributed to the early growth and development of biomolecular NMR, and our brief personal account certainly omits many important milestones.

Asunto(s)

RESUMEN

Nuclear magnetic resonance (NMR) spectroscopy has been instrumental during the past two decades in providing high-resolution structures of protein complexes. It has been the method of choice for determining the structure of dynamic protein complexes, which are typically recalcitrant to other structural techniques. Until recently, NMR spectroscopy has yielded structures of small or medium-sized protein complexes, up to approximately 30-40 kDa. Major breakthroughs during the past decade, especially in isotope-labeling techniques, have enabled NMR characterization of large protein systems with molecular weights of hundreds of kDa. This has provided unique insights into the binding, dynamic, and allosteric properties of large systems. Notably, there is now a slowly but steadily growing list of large, dynamic protein complexes whose atomic structure has been determined by NMR. Many of these complexes are characterized by a high degree of flexibility and, thus, their structures could not have been obtained using other structural methods. Especially in the field of molecular chaperones, NMR has recently provided the first-ever high-resolution structures of their complexes with unfolded proteins. Further technological advances will establish NMR as the primary tool for obtaining atomic structures of challenging systems with even higher complexity.

Asunto(s)