Untargeted metabolomics analysis of four date palm (Phoenix dactylifera L.) cultivars using MS and NMR.

Since ancient times, the inhabitants of dry areas have depended on the date palm (Phoenix dactylifera L.) as a staple food and means of economic security. For example, dates have been a staple diet for the inhabitants of the Arabian Peninsula and Sahara Desert in North Africa for millennia and the local culture is rich in knowledge and experience with the benefits of dates, suggesting that dates contain many substances essential for the human body. Madinah dates are considered one of the most important types of dates in the Arabian Peninsula, with Ajwa being one of the most famous types and grown only in Madinah, Saudi Arabia. Date seeds are traditionally used for animal feed, seed oil production, cosmetics, and as a coffee substitute. Phytochemical compounds that have been detected in date fruits and date seeds include phenolic acids, carotenoids, and flavonoids. Phenolic acids are the most prevalent bioactive constituents that contribute to the antioxidant activity of date fruits. The bioactive properties of these phytochemicals are believed to promote human health by reducing the risk of diseases such as chronic inflammation. Ajwa dates especially are thought to have superior bioactivity properties. To investigate these claims, in this study, we compare the metabolic profiles of Ajwa with different types of dates collected from Saudi Arabia and Tunisia. We show by UHPLC-MS that date seeds contain several classes of flavonoids, phenolic acids, and amino acid derivatives, including citric acid, malic acid, lactic acid, and hydroxyadipic acid. Additionally, GC-MS profiling showed that date seeds are richer in metabolite classes, such as hydrocinnamic acids (caffeic, ferulic and sinapic acids), than flesh samples. Deglet N fruit extract (minimum inhibitory concentration: 27 MIC/µM) and Sukkari fruit extract (IC50: 479 ± 0.58µg /mL) have higher levels of antibacterial and antioxidative activity than Ajwa fruits. However, the seed analysis showed that seed extracts have better bioactivity effects than fruit extracts. Specifically, Ajwa extract showed the best MIC and strongest ABTS radical-scavenging activity among examined seed extracts (minimum inhibitory concentration: 20 µM; IC50: 54 ± 3.61µg /mL). Our assays are a starting point for more advanced in vitro antibacterial models and investigation into the specific molecules that are responsible for the antioxidative and anti-bacterial activities of dates.

Lipoic Acid Restores Binding of Zinc Ions to Human Serum Albumin.

Human serum albumin (HSA) is the main zinc(II) carrier in blood plasma. The HSA site with the strongest affinity for zinc(II), multi-metal binding site A, is disrupted by the presence of fatty acids (FAs). Therefore, the FA concentration in the blood influences zinc distribution, which may affect both normal physiological processes and a range of diseases. Based on the current knowledge of HSA's structure and its coordination chemistry with zinc(II), we investigated zinc interactions and the effect of various FAs, including lipoic acid (LA), on the protein structure, stability, and zinc(II) binding. We combined NMR experiments and isothermal titration calorimetry to examine zinc(II) binding to HSA at a sub-atomic level in a quantitative manner as well as the effect of FAs. Free HSA results indicate the existence of one high-affinity zinc(II) binding site and multiple low-affinity sites. Upon the binding of FAs to HSA, we observed a range of behaviors in terms of zinc(II) affinity, depending on the type of FA. With FAs that disrupt zinc binding, the addition of LA restores HSA's affinity for zinc ions to the levels seen with free defatted HSA, indicating the possible mechanism of LA, which is effective in the treatment of diabetes and cardiovascular diseases.

Thymosin ß4 Is an Endogenous Iron Chelator and Molecular Switcher of Ferroptosis.

Thymosin ß4 (Tß4) was extracted forty years agofrom calf thymus. Since then, it has been identified as a G-actin binding protein involved in blood clotting, tissue regeneration, angiogenesis, and anti-inflammatory processes. Tß4 has also been implicated in tumor metastasis and neurodegeneration. However, the precise roles and mechanism(s) of action of Tß4 in these processes remain largely unknown, with the binding of the G-actin protein being insufficient to explain these multi-actions. Here we identify for the first time the important role of Tß4 mechanism in ferroptosis, an iron-dependent form of cell death, which leads to neurodegeneration and somehow protects cancer cells against cell death. Specifically, we demonstrate four iron2+ and iron3+ binding regions along the peptide and show that the presence of Tß4 in cell growing medium inhibits erastin and glutamate-induced ferroptosis in the macrophage cell line. Moreover, Tß4 increases the expression of oxidative stress-related genes, namely BAX, hem oxygenase-1, heat shock protein 70 and thioredoxin reductase 1, which are downregulated during ferroptosis. We state the hypothesis that Tß4 is an endogenous iron chelator and take part in iron homeostasis in the ferroptosis process. We discuss the literature data of parallel involvement of Tß4 and ferroptosis in different human pathologies, mainly cancer and neurodegeneration. Our findings confronted with literature data show that controlled Tß4 release could command on/off switching of ferroptosis and may provide novel therapeutic opportunities in cancer and tissue degeneration pathologies.

The robust NMR toolbox for metabolomics.

Here, we implemented and validated a suite of selective and non-selective CPMG-filtered 1D and 2D TOCSY/HSQC experiments for metabolomics research. They facilitated the unambiguous identification of metabolites embedded in broad lipid and protein signals. The 2D spectra improved non-targeted analysis by removing the background broad signals of macromolecules.

NMR-based metabolomics with enhanced sensitivity.

NMR-based metabolomics, which emerged along with mass spectrometry techniques, is the preferred method for studying metabolites in medical research and food industries. However, NMR techniques suffer from inherently low sensitivity, regardless of their superior reproducibility. To overcome this, we made two beneficial modifications: we detuned the probe to reach a position called "Spin Noise Tuning Optimum" (SNTO), and we replaced the conventional cylindrical 5 mm NMR tube with an electric field component-optimized shaped tube. We found that concerted use of both modifications can increase the sensitivity (signal to noise ratio per unit volume) and detection of metabolites and decrease the measurement time by order of magnitude. In this study, we demonstrate and discuss the achieved signal enhancement of metabolites on model non-human (bovine serum, amino acid standard mixture) and human urine samples.

Rapid nuclear magnetic resonance data acquisition with improved resolution and sensitivity for high-throughput metabolomic analysis.

Nuclear magnetic resonance (NMR)-based metabolomics has witnessed rapid advancements in recent years with the continuous development of new methods to enhance the sensitivity, resolution, and speed of data acquisition. Some of the approaches were earlier used for peptide and protein resonance assignments and have now been adapted to metabolomics. At the same time, new NMR methods involving novel data acquisition techniques, suited particularly for high-throughput analysis in metabolomics, have been developed. In this review, we focus on the different sampling strategies or data acquisition methods that have been developed in our laboratory and other groups to acquire NMR spectra rapidly with high sensitivity and resolution for metabolomics. In particular, we focus on the use of multiple receivers, phase modulation NMR spectroscopy, and fast-pulsing methods for identification and assignments of metabolites.

NMR as a "Gold Standard" Method in Drug Design and Discovery.

Studying disease models at the molecular level is vital for drug development in order to improve treatment and prevent a wide range of human pathologies. Microbial infections are still a major challenge because pathogens rapidly and continually evolve developing drug resistance. Cancer cells also change genetically, and current therapeutic techniques may be (or may become) ineffective in many cases. The pathology of many neurological diseases remains an enigma, and the exact etiology and underlying mechanisms are still largely unknown. Viral infections spread and develop much more quickly than does the corresponding research needed to prevent and combat these infections; the present and most relevant outbreak of SARS-CoV-2, which originated in Wuhan, China, illustrates the critical and immediate need to improve drug design and development techniques. Modern day drug discovery is a time-consuming, expensive process. Each new drug takes in excess of 10 years to develop and costs on average more than a billion US dollars. This demonstrates the need of a complete redesign or novel strategies. Nuclear Magnetic Resonance (NMR) has played a critical role in drug discovery ever since its introduction several decades ago. In just three decades, NMR has become a "gold standard" platform technology in medical and pharmacology studies. In this review, we present the major applications of NMR spectroscopy in medical drug discovery and development. The basic concepts, theories, and applications of the most commonly used NMR techniques are presented. We also summarize the advantages and limitations of the primary NMR methods in drug development.

Rapid NMR assignments of intrinsically disordered proteins using two-dimensional 13C-detection based experiments.

An approach for rapid backbone resonance assignments in proteins using only two 2D NMR experiments is presented. The new method involves a combination of high-resolution 13Cα-detected NMR experiments and selective unlabeling of amino acid residues. The 13C detected 2D hNCA and 2D hNcoCA spectra of a uniformly labeled sample of the protein are analysed in concert with the 2D hNCA spectrum obtained for a selectively unlabeled sample. The combinatorial set of amino acid residues for selective unlabeling is chosen optimally to maximize the assignments. The method is useful for rapid assignment of proteins with low stability such as intrinsically disordered proteins and is applicable to deuterated proteins. This approach helped in assignments of 14.5 kDa human α-synuclein during the course of its aggregation.

Enhancement of the Nuclear Spin Noise Signal Using Wavelet Transform.

Spin noise spectroscopy has attracted considerable attention recently owing partly to intrinsic interest in the phenomenon and partly to its significant application potential. Here, we address the inherent problem of low sensitivity of nuclear spin noise and examine the utility of wavelet transform to mitigate this problem by distinguishing real peaks from the noise contaminated data. Suppression of the random circuit noise and the consequent enhancement of the correlated nuclear spin noise signal have been demonstrated with discrete wavelet transform. Spectra of both 1 H and 13 C nuclear spins have been considered and significant signal enhancements in both the cases have been observed. A detailed analysis of several possible wavelet, thresholding and decomposition solutions have been made to obtain the optimum condition for signal enhancement. It is observed that the application of wavelet transform leaves the spin noise signal line shape essentially unchanged, which is an advantage for several applications involving spin noise spectra.

Spin-Noise-Detected Two-Dimensional Nuclear Magnetic Resonance at Triple Sensitivity.

A major breakthrough in speed and sensitivity of 2 D spin-noise-detected NMR is achieved owing to a new acquisition and processing scheme called "double block usage" (DBU) that utilizes each recorded noise block in two independent cross-correlations. The mixing, evolution, and acquisition periods are repeated head-to-tail without any recovery delays and well-known building blocks of multidimensional NMR (constant-time evolution and quadrature detection in the indirect dimension as well as pulsed field gradients) provide further enhancement and artifact suppression. Modified timing of the receiver electronics eliminates spurious random excitation. We achieve a threefold sensitivity increase over the original snHMQC (spin-noise-detected heteronuclear multiple quantum correlation) experiment (K. Chandra et al., J. Phys. Chem. Lett. 2013, 4, 3853) and demonstrate the feasibility of spin-noise-detected long-range correlation.

SOFAST-HMQC-an efficient tool for metabolomics.

Nuclear magnetic resonance (NMR)-based metabolomics relies mostly on 1D NMR; however, the technique is limited by overlap of the signals from the metabolites. In order to circumvent this problem, 2D 1H-13C correlation spectroscopy techniques are often used. However owing to poorer natural abundance and gyromagnetic ratio of 13C, the acquisition time for 2D 1H-13C heteronuclear single quantum coherence spectroscopy (HSQC) is long. This makes it almost impossible to be used in high throughput study. We have reported the application of selective optimized flip angle short transient (SOFAST) technique coupled to heteronuclear multiple quantum correlation (HMQC) along with nonlinear sampling (NUS) in urine and serum samples. This technique takes sevenfold less experimental time than the conventional 1H-13C HSQC experiment with retention of almost all molecular information. Hence, this can be used for high throughput study. Graphical abstract SOFAST-HMQC is a two-dimensional NMR technique that significantly decreases experimental time without loss of information. This technique is applied in complex biofluid samples that are used for high throughput metabolomics studies and shows promise of better information recovery than conventional two-dimensional NMR technique in shorter time.

Super-Resolved Nuclear Magnetic Resonance Spectroscopy.

We present a novel method that breaks the resolution barrier in nuclear magnetic resonance (NMR) spectroscopy, allowing one to accurately estimate the chemical shift values of highly overlapping or broadened peaks. This problem is routinely encountered in NMR when peaks have large linewidths due to rapidly decaying signals, hindering its application. We address this problem based on the notion of finite-rate-of-innovation (FRI) sampling, which is based on the premise that signals such as the NMR signal, can be accurately reconstructed using fewer measurements than that required by existing approaches. The FRI approach leads to super-resolution, beyond the limits of contemporary NMR techniques. Using this method, we could measure for the first time small changes in chemical shifts during the formation of a Gold nanorod-protein complex, facilitating the quantification of the strength of such interactions. The method thus opens up new possibilities for the application and acceleration of multidimensional NMR spectroscopy across a wide range of systems.

Envisaging the Structural Elevation in the Early Event of Oligomerization of Disordered Amyloid ß Peptide.

In Alzheimer's disease (AD), amyloid ß (Aß) protein plays a detrimental role in neuronal injury and death. Recent in vitro and in vivo studies suggest that soluble oligomers of the Aß peptide are neurotoxic. Structural properties of the oligomeric assembly, however, are largely unknown. Our present investigation established that the 40-residue-long Aß peptide (Aß40) became more helical, ordered, and compact in the oligomeric state, and both the helical and ß-sheet components were found to increase significantly in the early event of oligomerization. The band-selective two-dimensional NMR analysis suggested that majority of the residues from sequence 12 to 22 gained a higher-ordered secondary structure in the oligomeric condition. The presence of a significant amount of helical conformation was confirmed by Raman bands at 1650 and 1336 cm-1. Other residues remained mostly in the extended polyproline II (PPII) and less compact ß-conformation space. In the event of maturation of the oligomers into an amyloid fiber, both the helical content and the PPII-like structural components declined and ∼72% residues attained a compact ß-sheet structure. Interestingly, however, some residues remained in the collagen triple helix/extended 2.51-helix conformation as evidenced by the amide III Raman signature band at 1272 cm-1. Molecular dynamics analysis using an optimized potential for liquid simulation force field with the peptide monomer indicated that some of the residues may have preferences for helical conformation and this possibly contributed in the event of oligomer formation, which eventually became a ß-sheet-rich amyloid fiber.

Rapid identification of amino acid types in proteins using phase modulated 2D HN(CACB) and 2D HN(COCACB).

We present a simple approach to rapidly identify amino acid types in proteins from a 2D spectrum. The method is based on the fact that (13)C(ß) chemical shifts of different amino acid types fall in distinct spectral regions. By evolving the (13)C chemical shifts in the conventional HNCACB or HN(CO)CACB type experiment for a single specified delay period, the phase of the cross peaks of different amino acid residues are modulated depending on their (13)C(ß) shift values. Following this specified evolution period, the 2D HN projections of these experiments are acquired. The (13)C evolution period can be chosen such that all residues belonging to a given set of amino acid types have the same phase pattern (positive or negative) facilitating their identification. This approach does not require the preparation of any additional samples, involves the analysis of 2D [(15)N-(1)H] HSQC-type spectra obtained from the routinely used triple resonance experiments with minor modifications, and is applicable to deuterated proteins. The method will be useful for quick assignment of signals that shift during ligand binding or in combination with selective labeling/unlabeling approaches for identification of amino acid types to aid the sequential assignment process.

Solution NMR and molecular dynamics reveal a persistent alpha helix within the dynamic region of PsbQ from photosystem II of higher plants.

The extrinsic proteins of photosystem II of higher plants and green algae PsbO, PsbP, PsbQ, and PsbR are essential for stable oxygen production in the oxygen evolving center. In the available X-ray crystallographic structure of higher plant PsbQ residues S14-Y33 are missing. Building on the backbone NMR assignment of PsbQ, which includes this "missing link", we report the extended resonance assignment including side chain atoms. Based on nuclear Overhauser effect spectra a high resolution solution structure of PsbQ with a backbone RMSD of 0.81 Å was obtained from torsion angle dynamics. Within the N-terminal residues 1-45 the solution structure deviates significantly from the X-ray crystallographic one, while the four-helix bundle core found previously is confirmed. A short α-helix is observed in the solution structure at the location where a ß-strand had been proposed in the earlier crystallographic study. NMR relaxation data and unrestrained molecular dynamics simulations corroborate that the N-terminal region behaves as a flexible tail with a persistent short local helical secondary structure, while no indications of forming a ß-strand are found.

An ultrastable conjugate of silver nanoparticles and protein formed through weak interactions.

In recent years, silver nanoparticles (AgNPs) have attracted significant attention owing to their unique physicochemical, optical, conductive and antimicrobial properties. One of the properties of AgNPs which is crucial for all applications is their stability. In the present study we unravel a mechanism through which silver nanoparticles are rendered ultrastable in an aqueous solution in complex with the protein ubiquitin (Ubq). This involves a dynamic and reversible association and dissociation of ubiquitin from the surface of AgNP. The exchange occurs at a rate much greater than 25 s(-1) implying a residence time of <40 ms for the protein. The AgNP-Ubq complex remains stable for months due to steric stabilization over a wide pH range compared to unconjugated AgNPs. NMR studies reveal that the protein molecules bind reversibly to AgNP with an approximate dissociation constant of 55 µM and undergo fast exchange. At pH > 4 the positively charged surface of the protein comes in contact with the citrate capped AgNP surface. Further, NMR relaxation-based experiments suggest that in addition to the dynamic exchange, a conformational rearrangement of the protein takes place upon binding to AgNP. The ultrastability of the AgNP-Ubq complex was found to be useful for its anti-microbial activity, which allowed the recycling of this complex multiple times without the loss of stability. Altogether, the study provides new insights into the mechanism of protein-silver nanoparticle interactions and opens up new avenues for its application in a wide range of systems.

Quantitative metabolic profiling of NMR spectral signatures of branched chain amino acids in blood serum.

Branched Chain Amino Acids (BCAAs) are related to different aspects of diseases like pathogenesis, diagnosis and even prognosis. While in some diseases, levels of all the BCAAs are perturbed; in some cases, perturbation occurs in one or two while the rest remain unaltered. In case of ischemic heart disease, there is an enhanced level of plasma leucine and isoleucine but valine level remains unaltered. In 'Hypervalinemia', valine is elevated in serum and urine, but not leucine and isoleucine. Therefore, identification of these metabolites and profiling of individual BCAA in a quantitative manner in body-fluid like blood plasma/serum have long been in demand. (1)H NMR resonances of the BCAAs overlap with each other which complicates quantification of individual BCAAs. Further, the situation is limited by the overlap of broad resonances of lipoprotein with the resonances of BCAAs. The widely used commercially available kits cannot differentially estimate the BCAAs. Here, we have achieved proper identification and characterization of these BCAAs in serum in a quantitative manner employing a Nuclear Magnetic Resonance-based technique namely T2-edited Correlation Spectroscopy (COSY). This approach can easily be extended to other body fluids like bile, follicular fluids, saliva, etc.

Resonance assignment of PsbP: an extrinsic protein from photosystem II of Spinacia oleracea.

PsbP (23 kDa) is an extrinsic eukaryotic protein of photosystem II found in the thylakoid membrane of higher plants and green algae. It has been proven to be indispensable for proper functioning of the oxygen evolving complex. By interaction with other extrinsic proteins (PsbQ, PsbO and PsbR), it modulates the concentration of two cofactors of the water splitting reaction, Ca(2+) and Cl(-). The crystallographic structure of PsbP from Spinacia oleracea lacks the N-terminal part as well as two inner regions which were modelled as loops. Those unresolved parts are believed to be functionally crucial for the binding of PsbP to the thylakoid membrane. In this NMR study we report (1)H, (15)N and (13)C resonance assignments of the backbone and side chain atoms of the PsbP protein. Based on these data, an estimate of the secondary structure has been made. The structural motifs found fit the resolved parts of the crystallographic structure very well. In addition, the complete assignment set provides preliminary insight into the dynamic regions.

Liaison between myristoylation and cryptic EF-hand motif confers Ca(2+) sensitivity to neuronal calcium sensor-1.

Many members of the neuronal calcium sensor (NCS) protein family have a striking coexistence of two characteristics, that is, N-myristoylation and the cryptic EF-1 motif. We investigated the rationale behind this correlation in neuronal calcium sensor-1 (NCS-1) by restoring Ca(2+) binding ability of the disabled EF-1 loop by appropriate mutations. The concurrence of canonical EF-1 and N-myristoylation considerably decreased the overall Ca(2+) affinity, conformational flexibility, and functional activation of downstream effecter molecules (i.e., PI4Kß). Of a particular note, Ca(2+) induced conformational change (which is the first premise for a CaBP to be considered as sensor) is considerably reduced in myristoylated proteins in which Ca(2+)-binding to EF-1 is restored. Moreover, Ca(2+), which otherwise augments the enzymatic activity of PI4Kß (modulated by NCS-1), leads to a further decline in the modulated PI4Kß activity by myristoylated mutants (with canonical EF-1) pointing toward a loss of Ca(2+) signaling and specificity at the structural as well as functional levels. This study establishes the presence of the strong liaison between myristoylation and cryptic EF-1 in NCS-1. Breaking this liaison results in the failure of Ca(2+) specific signal transduction to downstream effecter molecules despite Ca(2+) binding. Thus, the EF-1 disability is a prerequisite in order to append myristoylation signaling while preserving structural robustness and Ca(2+) sensitivity/specificity in NCS-1.

Rapid characterization of molecular diffusion by NMR spectroscopy.

An NMR-based approach for rapid characterization of translational diffusion of molecules has been developed. Unlike the conventional method of acquiring a series of 2D (13)C and (1)H spectra, the proposed approach involves a single 2D NMR spectrum, which can be acquired in minutes. Using this method, it was possible to detect the presence of intermediate oligomeric species of diphenylalanine in solution during the process of its self-assembly to form nanotubular structures.