RESUMEN
It is advantageous to investigate milli-to-micro-second conformational exchange data contained in the solution NMR protein relaxation data other than 15N nuclei. Not only does one search under another lamp post, one also looks at dynamics at other time scales. The HSQC-ROESY 1HN relaxation dispersion experiment for amide protons as introduced by Ishima, et al (1998). J. Am. Soc. 120, 10534-10542, is such an experiment, but has by the authors been advised to only be used for perdeuterated proteins to avoid complication with the 1H-1H multiple-spin effects. This is regretful, since not all proteins can be perdeuterated. Here we analyze in detail the 1HN relaxation terms for this experiment for a fully proteated protein. Indeed, the 1HN relaxation theory is in this case complex and includes dipolar-dipolar relaxation interference and TOCSY transfers. With simulate both of these effects and show that the interference can be exploited for detecting exchange broadening. The TOCSY effect is shown to minor, and when it is not, a solution is provided. We apply the HSQC-ROESY experiment, with a small modification to suppress ROESY crosspeaks, to a 7 kDa GB1 protein that is just 15N and 13C labeled. At 10 °C we cannot detect any conformational exchange broadening: the 1HN R2 relaxation rates with 1.357 kHz spinlock field not larger than those recorded with a 12.136 kHz spinlock field. This means that there is no exchange broadening that can be differentially suppressed with the applied fields. Either there is no broadening, or the broadening is effectively suppressed by all fields, or the broadening cannot be suppressed by either of the fields. While initially this seems to be a disappointing result, we feel that this work establishes that the HSQC-ROESY experiment is very robust. It can indeed be utilized for proteated proteins upto about 30 kDa. This could be opening the study the milli-microsecond conformational dynamics as reported by 1HN exchange broadening for many more proteins.
Asunto(s)
Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Proteínas , Isótopos de Nitrógeno/química , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Algoritmos , Conformación Proteica , ProtonesRESUMEN
Metronidazole is a prospective hyperpolarized MRI contrast agent with potential hypoxia sensing utility for applications in cancer, stroke, neurodegenerative diseases, etc. We demonstrate a pilot procedure for production of â¼30 mM hyperpolarized [15N3]metronidazole in aqueous media by using a phase-separated SABRE-SHEATH hyperpolarization method, with nitrogen-15 polarization exceeding 2.2% on all three 15N sites achieved in less than 2 min. The 15N polarization T1 of â¼12 min is reported for the 15NO2 group at the clinically relevant field of 1.4 T in the aqueous phase, demonstrating a remarkably long lifetime of the hyperpolarized state. The produced aqueous solution of [15N3]metronidazole that contained only â¼100 µM of residual Ir was deemed biocompatible via validation through the MTT colorimetric test for assessing cell metabolic activity using human embryotic kidney HEK293T cells. This low-cost and ultrafast hyperpolarization procedure represents a major advance for the production of a biocompatible HP [15N3]metronidazole (and potentially other hyperpolarized drugs) formulation for MRI sensing applications.
Asunto(s)
Metronidazol , Agua , Humanos , Metronidazol/química , Metronidazol/farmacología , Células HEK293 , Agua/química , Antibacterianos/química , Antibacterianos/farmacología , Hidrógeno/química , Isótopos de Nitrógeno/química , Imagen por Resonancia Magnética/métodos , Medios de Contraste/químicaRESUMEN
Magnetic resonance with hyperpolarized contrast agents is one of the most powerful and noninvasive imaging platforms capable for investigating in vivo metabolism. While most of the utilized hyperpolarized agents are based on 13C nuclei, a milestone advance in this area is the emergence of 15N hyperpolarized contrast agents. Currently, the reported 15N hyperpolarized agents mainly utilize the dissolution dynamic nuclear polarization (d-DNP) protocol. The parahydrogen enhanced 15N probes have proven to be elusive and have been tested almost exclusively in organic solvents. Herein, we designed a reaction based reactive oxygen sensor 15N-boronobenzyl-2-styrylpyridinium (15N-BBSP) which can be hyperpolarized with para-hydrogen. Reactive oxygen species plays a vital role as one of the essential intracellular signalling molecules. Disturbance of the H2O2 level usually represents a hallmark of pathophysiological conditions. This H2O2 probe exhibited rapid responsiveness toward H2O2 and offered spectrally resolvable chemical shifts. We also provide strategies to bring the newly developed probe from the organic reaction solution into a biocompatible injection buffer and demonstrate the feasibility of in vivo 15N signal detection. The present work manifests its great potential not only for reaction based reactive sensing probes but also promises to serve as a platform to develop other contrast agents.
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Hidrógeno , Compuestos de Piridinio , Especies Reactivas de Oxígeno , Compuestos de Piridinio/química , Hidrógeno/química , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/análisis , Animales , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/análisis , Isótopos de Nitrógeno/química , Ratones , Proyectos Piloto , Estructura Molecular , Medios de Contraste/químicaRESUMEN
Here we report the efficient synthetic access to 13C/15N-labelled pseudouridine phosphoramidites, which were incorporated into a binary H/ACA box guide RNA/product complex comprising 77 nucleotides (nts) in total and into a 75â nt E. coli tRNAGly. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo-enzymatic synthesis. 13C/15N labelled uracils were produced via chemical synthesis and enzymatically converted to pseudouridine 5'-monophosphate (ΨMP) by using YeiN, a Ψ-5'-monophosphate C-glycosidase. Removal of the 5'-phosphate group yielded the desired pseudouridine nucleoside (Ψ), which was transformed into a phosphoramidite building suitable for RNA solid phase synthesis. A Ψ -building block carrying both a 13C and a 15N label was incorporated into a product RNA and the complex formation with a 63â nt H/ACA box RNA could be observed via NMR. Furthermore, the SI labelled pseudouridine building block was used to determine imino proton bulk water exchange rates of a 75â nt E. coli tRNAGly CCmnm5U, identifying the TΨC-loop 5-methyluridine as a modifier of the exchange rates. The efficient synthetic access to SI-labelled Ψ building blocks will allow the solution and solid-state NMR spectroscopic studies of Ψ containing RNAs and will facilitate the mass spectrometric analysis of Ψ-modified nucleic acids.
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Escherichia coli , Marcaje Isotópico , Isótopos de Nitrógeno , Compuestos Organofosforados , Seudouridina , Seudouridina/química , Compuestos Organofosforados/química , Isótopos de Nitrógeno/química , Marcaje Isotópico/métodos , ARN/química , Isótopos de Carbono/química , Espectroscopía de Resonancia Magnética/métodosRESUMEN
Crystallization is a widely used purification technique in the manufacture of active pharmaceutical ingredients (APIs) and precursor molecules. However, when impurities and desired compounds have similar molecular structures, separation by crystallization may become challenging. In such cases, some impurities may form crystalline solid solutions with the desired product during recrystallization. Understanding the molecular structure of these recrystallized solid solutions is crucial to devise methods for effective purification. Unfortunately, there are limited analytical techniques that provide insights into the molecular structure or spatial distribution of impurities that are incorporated within recrystallized products. In this study, we investigated model solid solutions formed by recrystallizing salicylic acid (SA) in the presence of anthranilic acid (AA). These two molecules are known to form crystalline solid solutions due to their similar molecular structures. To overcome challenges associated with the long 1H longitudinal relaxation times (T1(1H)) of SA and AA, we employed dynamic nuclear polarization (DNP) and 15N isotope enrichment to enable solid-state NMR experiments. Results of solid-state NMR experiments and DFT calculations revealed that SA and AA are homogeneously alloyed as a solid solution. Heteronuclear correlation (HETCOR) experiments and plane-wave DFT structural models provide further evidence of the molecular-level interactions between SA and AA. This research provides valuable insights into the molecular structure of recrystallized solid solutions, contributing to the development of effective purification strategies and an understanding of the physicochemical properties of solid solutions.
Asunto(s)
Isótopos de Carbono , Cristalización , Espectroscopía de Resonancia Magnética , Isótopos de Nitrógeno , Ácido Salicílico , ortoaminobenzoatos , Espectroscopía de Resonancia Magnética/métodos , Ácido Salicílico/química , Cristalización/métodos , Isótopos de Nitrógeno/química , ortoaminobenzoatos/química , Isótopos de Carbono/química , Soluciones/química , Estructura MolecularRESUMEN
The present work investigates the potential for enhancing the NMR signals of DNA nucleobases by parahydrogen-based hyperpolarization. Signal amplification by reversible exchange (SABRE) and SABRE in Shield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) of selected DNA nucleobases is demonstrated with the enhancement (ε) of 1H, 15N, and/or 13C spins in 3-methyladenine, cytosine, and 6-O-guanine. Solutions of the standard SABRE homogenous catalyst Ir(1,5-cyclooctadeine)(1,3-bis(2,4,6-trimethylphenyl)imidazolium)Cl ("IrIMes") and a given nucleobase in deuterated ethanol/water solutions yielded low 1H ε values (≤10), likely reflecting weak catalyst binding. However, we achieved natural-abundance enhancement of 15N signals for 3-methyladenine of ~3300 and ~1900 for the imidazole ring nitrogen atoms. 1H and 15N 3-methyladenine studies revealed that methylation of adenine affords preferential binding of the imidazole ring over the pyrimidine ring. Interestingly, signal enhancements (ε~240) of both 15N atoms for doubly labelled cytosine reveal the preferential binding of specific tautomer(s), thus giving insight into the matching of polarization-transfer and tautomerization time scales. 13C enhancements of up to nearly 50-fold were also obtained for this cytosine isotopomer. These efforts may enable the future investigation of processes underlying cellular function and/or dysfunction, including how DNA nucleobase tautomerization influences mismatching in base-pairing.
Asunto(s)
Imidazoles , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Isótopos de Nitrógeno/química , ADNRESUMEN
Magnetic resonance imaging (MRI) provides unique information about the internal structure and function of living organisms in a non-invasive way. The use of conventional proton MRI for the observation of real-time metabolism is hampered by the dominant signals of water and fat, which are abundant in living organisms. Heteronuclear MRI in conjunction with the hyperpolarization methods does not encounter this issue. In this work, we polarized 15N nuclei of [15N1]fampridine (a drug used for the treatment of multiple sclerosis) to the level of 4% in nuclear magnetic resonance (NMR) experiments and 0.7% in MRI studies using spin-lock-induced crossing combined with signal amplification by reversible exchange. Consequently, three-dimensional 15N MRI of the hyperpolarized 15N-labeled drug was acquired in 0.1 s with a signal-to-noise ratio of 70. In addition, the NMR signal enhancements for 15N-enriched fampridine and fampridine with a natural abundance of 15N nuclei were compared and an explanation for their difference was proposed.
Asunto(s)
Imagen por Resonancia Magnética , Protones , Espectroscopía de Resonancia Magnética/métodos , Imagen por Resonancia Magnética/métodos , Isótopos de Nitrógeno/químicaRESUMEN
The impacts of enhanced nitrogen (N) deposition on the global forest carbon (C) sink and other ecosystem services may depend on whether N is deposited in reduced (mainly as ammonium) or oxidized forms (mainly as nitrate) and the subsequent fate of each. However, the fates of the two key reactive N forms and their contributions to forest C sinks are unclear. Here, we analyze results from 13 ecosystem-scale paired 15N-labelling experiments in temperate, subtropical, and tropical forests. Results show that total ecosystem N retention is similar for ammonium and nitrate, but plants take up more labelled nitrate ([Formula: see text]%) ([Formula: see text]) than ammonium ([Formula: see text]%) while soils retain more ammonium ([Formula: see text]%) than nitrate ([Formula: see text]%). We estimate that the N deposition-induced C sink in forests in the 2010s is [Formula: see text] Pg C yr-1, higher than previous estimates because of a larger role for oxidized N and greater rates of global N deposition.
Asunto(s)
Compuestos de Amonio/análisis , Secuestro de Carbono/fisiología , Restauración y Remediación Ambiental , Bosques , Nitratos/análisis , Árboles/metabolismo , Ambiente , Isótopos de Nitrógeno/química , Óxidos de Nitrógeno/análisis , Suelo/químicaRESUMEN
Compound-specific stable isotope analysis (CSIA) is a unique analytical technique for determining small variations in isotope ratios of light isotopes in analytes from complex mixtures. A problem of CSIA using gas chromatography (GC) and liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) is that any structural information of the analytes is lost due to the processes involved in determining the isotope ratio. To obtain the isotopic composition of, for example, carbon from organic compounds, all carbon in each analyte is quantitatively converted to CO2. For GC-IRMS, open split GC-IRMS-MS couplings have been described that allow additional acquisition of structural information of analytes and interferences. Structural analysis using LC-IRMS is more difficult and requires additional technical and instrumental efforts. In this study, LC was combined for the first time with simultaneous analysis by IRMS and high-resolution mass spectrometry (HRMS), enabling the direct identification of unknown or coeluting species. We have thoroughly investigated and optimized the coupling and showed how technical problems, arising from instrumental conditions, can be overcome. To this end, it was successfully demonstrated that a consistent split ratio between IRMS and HRMS could be obtained using a variable postcolumn flow splitter. This coupling provided reproducible results in terms of resulting peak areas, isotope values, and retention time differences for the two mass spectrometer systems. To demonstrate the applicability of the coupling, we chose to address an important question regarding the purity of international isotope standards. In this context, we were able to confirm that the USGS41 reference material indeed contains substantial amounts of pyroglutamic acid as suggested previously in the literature. Moreover, the replacement material, USGS41a, still has significant amounts of pyroglutamic acid as impurity, rendering some caution necessary when using this material for isotopic calibration.
Asunto(s)
Isótopos de Carbono , Isótopos de Carbono/análisis , Cromatografía Liquida/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Espectrometría de Masas/métodos , Isótopos de Nitrógeno/químicaRESUMEN
Structural characterization of the SARS-CoV-2 full length nsp1 protein will be an essential tool for developing new target-directed antiviral drugs against SARS-CoV-2 and for further understanding of intra- and intermolecular interactions of this protein. As a first step in the NMR studies of the protein, we report the 1H, 13C and 15N resonance backbone assignment as well as the Cß of the apo form of the full-lengthSARS-CoV-2 nsp1 including the folded domain together with the flaking N- and C- terminal intrinsically disordered fragments. The 19.8 kD protein was characterized by high-resolution NMR. Validation of assignment have been done by using two different mutants, H81P and K129E/D48E as well as by amino acid specific experiments. According to the obtained assignment, the secondary structure of the folded domain in solution was almost identical to its previously published X-ray structure as well as another published secondary structure obtained by NMR, but some discrepancies have been detected. In the solution SARS-CoV-2 nsp1 exhibited disordered, flexible N- and C-termini with different dynamic characteristics. The short peptide in the beginning of the disordered C-terminal domain adopted two different conformations distinguishable on the NMR time scale. We propose that the disordered and folded nsp1 domains are not fully independent units but are rather involved in intramolecular interactions. Studies of the structure and dynamics of the SARS-CoV-2 mutant in solution are on-going and will provide important insights into the molecular mechanisms underlying these interactions.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , COVID-19/patología , COVID-19/virología , Espectroscopía de Resonancia Magnética con Carbono-13 , Humanos , Mutación , Isótopos de Nitrógeno/química , Estructura Secundaria de Proteína , Espectroscopía de Protones por Resonancia Magnética , SARS-CoV-2/aislamiento & purificación , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
RATIONALE: Lipid correction models use elemental carbon-to-nitrogen ratios to estimate the effect of lipids on δ13 C values and provide a fast and inexpensive alternative to chemically removing lipids. However, the performance of these models varies, especially in whole-body invertebrate samples. The generation of tissue-specific lipid correction models for American lobsters, both an ecologically and an economically important species in eastern North America, will aid ecological research of this species and our understanding of the function of these models in invertebrates. METHOD: We determined the δ13 C and δ15 N values before and after lipid extraction in muscle and digestive glands of juvenile and adult lobster. We assessed the performance of four commonly used models (nonlinear, linear, natural logarithm (LN) and generalized linear model (GLM)) at estimating lipid-free δ13 C values based on the non-lipid-extracted δ13 C values and elemental C:N ratios. The accuracy of model predictions was tested using paired t-tests, and the performance of the different models was compared using the Akaike information criterion score. RESULTS: Lipid correction models accurately estimated post-lipid-extraction δ13 C values in both tissues. The nonlinear model was the least accurate for both tissues. In muscle, the three other models performed well, and in digestive glands, the LN model provided the most accurate estimates throughout the range of C:N values. In both tissues, the GLM estimates were not independent of the post-lipid-extraction δ13 C values, thus reducing their transferability to other datasets. CONCLUSIONS: Whereas previous work found that whole-body models poorly estimated the effect of lipids in invertebrates, we show that tissue-specific lipid correction models can generate accurate and precise estimates of lipid-free δ13 C values in lobster. We suggest that the tissue-specific logarithmic models presented here are the preferred models for accounting for the effect of lipid on lobster isotope ratios.
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Isótopos de Carbono/química , Lípidos/química , Nephropidae/química , Animales , Isótopos de Carbono/metabolismo , Sistema Digestivo/química , Sistema Digestivo/metabolismo , Metabolismo de los Lípidos , Espectrometría de Masas , Músculos/química , Músculos/metabolismo , Nephropidae/metabolismo , Isótopos de Nitrógeno/química , Isótopos de Nitrógeno/metabolismo , Mariscos/análisisRESUMEN
Cellular DNA is composed of four canonical nucleosides (dA, dC, dG and T), which form two Watson-Crick base pairs. In addition, 5-methylcytosine (mdC) may be present. The methylation of dC to mdC is known to regulate transcriptional activity. Next to these five nucleosides, the genome, particularly of stem cells, contains three additional dC derivatives, which are formed by stepwise oxidation of the methyl group of mdC with the help of Tet enzymes. These are 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC). It is believed that fdC and cadC are converted back into dC, which establishes an epigenetic control cycle that starts with methylation of dC to mdC, followed by oxidation and removal of fdC and cadC. While fdC was shown to undergo intragenomic deformylation to give dC directly, a similar decarboxylation of cadC was postulated but not yet observed on the genomic level. By using metabolic labelling, we show here that cadC decarboxylates in several cell types, which confirms that both fdC and cadC are nucleosides that are directly converted back to dC within the genome by C-C bond cleavage.
Asunto(s)
ADN/metabolismo , Desoxicitidina/análogos & derivados , Genoma/fisiología , Animales , Células CHO , Cricetulus , ADN/química , Descarboxilación , Desoxicitidina/química , Desoxicitidina/metabolismo , Deuterio/química , Ratones , Isótopos de Nitrógeno/químicaRESUMEN
RATIONALE: Around the world biosecurity measures are being strengthened to prevent the spread of pests and diseases across national and international borders. Quarantine protocols that involve sample sterilisation have potential effects on sample integrity. The consequences of sterilisation methods such as gamma (γ)-irradiation on the elemental and chemical properties of biological samples have not been widely examined. METHODS: We tested the effect of γ-irradiation (50 kGy) on the stable carbon and nitrogen isotope compositions (δ13 C and δ15 N values) and elemental concentrations (C % and N %) of common biological samples (fish, plants and bulk soils). The analysis used a continuous flow system consisting of a Delta V Plus isotope ratio mass spectrometer connected with a Thermo Flash 1112 elemental analyser via a ConFlo IV interface. Results were compared using two one-sided tests (TOST) to test for statistical similarity between paired samples. RESULTS: There was no change in the δ15 N values or N % of γ-irradiated samples, and only small changes to the δ13 C values of consumers (range: 0.01 to 0.04), producers (-0.02 to 0.04) and sediments (0 to 0.07). The magnitude of change in δ13 C values was greatest at low carbon concentrations but appeared negligible when measured against replicated sample analysis and the combined analytical uncertainty (i.e., 0.10). The C % values of irradiated samples were higher for consumers (0.23%) and lower for producers and sediments (0.04% and 0.05%, respectively) which may have implications for certain types of biological material. CONCLUSIONS: Routine γ-irradiation has little effect on the stable carbon and nitrogen isotope compositions of common biological samples and marginal effects on carbon elemental concentrations. This is unlikely to warrant concerns since the observed difference is typically of a magnitude lower than other sources of potential uncertainty.
Asunto(s)
Bioaseguramiento , Isótopos de Carbono/análisis , Rayos gamma , Espectrometría de Masas/métodos , Isótopos de Nitrógeno/análisis , Animales , Isótopos de Carbono/química , Peces , Isótopos de Nitrógeno/química , Plantas/química , Plantas/efectos de la radiación , Suelo , EsterilizaciónRESUMEN
Carnivorous plants consume animals for mineral nutrients that enhance growth and reproduction in nutrient-poor environments. Here, we report that Triantha occidentalis (Tofieldiaceae) represents a previously overlooked carnivorous lineage that captures insects on sticky inflorescences. Field experiments, isotopic data, and mixing models demonstrate significant N transfer from prey to Triantha, with an estimated 64% of leaf N obtained from prey capture in previous years, comparable to levels inferred for the cooccurring round-leaved sundew, a recognized carnivore. N obtained via carnivory is exported from the inflorescence and developing fruits and may ultimately be transferred to next year's leaves. Glandular hairs on flowering stems secrete phosphatase, as seen in all carnivorous plants that directly digest prey. Triantha is unique among carnivorous plants in capturing prey solely with sticky traps adjacent to its flowers, contrary to theory. However, its glandular hairs capture only small insects, unlike the large bees and butterflies that act as pollinators, which may minimize the conflict between carnivory and pollination.
Asunto(s)
Alismatales/fisiología , Planta Carnívora/fisiología , Inflorescencia/fisiología , Isótopos de Nitrógeno/metabolismo , Animales , Drosophila/química , Ecosistema , Nitrógeno/metabolismo , Isótopos de Nitrógeno/químicaRESUMEN
The resolving power of solid-state nuclear magnetic resonance (NMR) crystallography depends heavily on the accuracy of computational predictions of NMR chemical shieldings of candidate structures, which are usually taken to be local minima in the potential energy. To test the limits of this approximation, we systematically study the importance of finite-temperature and quantum nuclear fluctuations for 1H, 13C, and 15N shieldings in polymorphs of three paradigmatic molecular crystals: benzene, glycine, and succinic acid. The effect of quantum fluctuations is comparable to the typical errors of shielding predictions for static nuclei with respect to experiments, and their inclusion improves the agreement with measurements, translating to more reliable assignment of the NMR spectra to the correct candidate structure. The use of integrated machine-learning models, trained on first-principles energies and shieldings, renders rigorous sampling of nuclear fluctuations affordable, setting a new standard for the calculations underlying NMR structure determinations.
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Benceno/química , Glicina/química , Ácido Succínico/química , Isótopos de Carbono/química , Cristalografía/métodos , Hidrógeno/química , Aprendizaje Automático , Espectroscopía de Resonancia Magnética , Isótopos de Nitrógeno/químicaRESUMEN
Determining which proteins are actively synthesized at a given point in time and extracting a representative sample for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) of a small sample of the proteins being synthesized in Arabidopsis plants or cell cultures, facilitating their click-chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met sites throughout protein amino acid sequences and correlation with independent studies of protein labelling with 15 N verified the data. We provide evidence that HPG-based BONCAT tags a better sample of nascent plant proteins than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that the AHA induction of Met metabolism and greater inhibition of cell growth rate than HPG probably limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for sampling, which plant proteins are being synthesized at a given time point and enriches a small portion of new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides samples were found to contain significantly fewer common post-translationally modified residues than the same proteins from whole plant extracts, providing evidence for age-related accumulation of post-translational modifications in plants.
Asunto(s)
Alquinos/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/aislamiento & purificación , Arabidopsis/química , Glicina/análogos & derivados , Proteómica/métodos , Alanina/análogos & derivados , Alanina/química , Arabidopsis/citología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ontología de Genes , Glicina/química , Espectrometría de Masas , Metionina/química , Metionina/metabolismo , Isótopos de Nitrógeno/química , Células Vegetales , Procesamiento Proteico-PostraduccionalRESUMEN
The amino-terminal-copper-and-nickel-binding (ATCUN) motif, a tripeptide sequence ending with a histidine, confers important functions to proteins and peptides. Few high-resolution studies have been performed on the ATCUN motifs of membrane-associated proteins and peptides, limiting our understanding of how they stabilize Cu2+/Ni2+ in membranes. Here, we leverage solid-state NMR to investigate metal-binding to piscidin-1 (P1), a host-defense peptide featuring F1F2H3 as its ATCUN motif. Bound to redox ions, P1 chemically and physically damages pathogenic cell membranes. We design 13C/15N correlation experiments to detect and assign the deprotonated nitrogens produced and/or shifted by Ni2+-binding. Occupying multiple chemical states in P1-apo, H3 and the neighboring H4 respond to metalation by populating only the τ-tautomer. H3, as a proximal histidine, directly coordinates the metal, compared to the distal H4. Density functional theory calculations reflect this noncanonical arrangement and point toward cation-π interactions between the F1/F2/H4 aromatic rings and metal. These structural findings, which are relevant to other ATCUN-containing membrane peptides, could help design new therapeutics and materials for use in the areas of drug-resistant bacteria, neurological disorders, and biomedical imaging.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Cobre/química , Proteínas de Peces/química , Níquel/química , Isótopos de Carbono/química , Cationes Bivalentes/química , Teoría Funcional de la Densidad , Espectroscopía de Resonancia Magnética , Isótopos de Nitrógeno/química , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The aggregation process of α-synuclein (α-syn) is substantial in the pathogenesis of Parkinson's disease. Indolinone derivatives are inhibitors of α-syn aggregates and can be used as PET-based radiotracers for imaging α-syn fibrils. However, no investigations on the metabolism of indolinone derivatives have been reported until now. In the present research, a 13C and 15N isotope labeling strategy was developed to synthesize compound [13C2,15N]-(Z)-1-(4-aminobenzyl)-3-((E)-(3-phenyl)allylidene)indolin-2-one (M0'), which was then used in a study of metabolism in hepatocytes. The metabolites were characterized using accurate mass and characteristic ion measurements. In the metabolic system, compound M0' was the main component (accounting for 97.5% of compound-related components) after incubation in hepatocytes for 3 h, which indicated that compound M0' possessed great metabolic stability. Seven metabolites have been successfully verified by UPLC/Q TOF MS in metabolic studies, including hydroxyl M0' (M1'), hydroxyl and methylated M0' (M2'), N-acetylated M0' (M3'), sulfate of hydroxyl M0' (M4'), the glucose conjugate of M0' (M5'), glucuronide conjugate of M0' (M6'), and glucuronide conjugate of hydroxyl M0' (M7'). The study on metabolism provides the important information to develop effective α-syn aggregate inhibitors and new PET-tracer-related indolinone derivatives.
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Espectrometría de Masas/métodos , Oxindoles/química , Isótopos de Carbono/química , Línea Celular , Cromatografía Líquida de Alta Presión/métodos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Marcaje Isotópico , Isótopos de Nitrógeno/químicaRESUMEN
Cytochrome c (cyt c) is widely used as a model protein to study (i) folding and stability aspects of the protein folding problem and (ii) structure-function relationship from the evolutionary point of view. Databases of cyts c now contain 285 cyt c sequences from different organisms. A sequence alignment of all these proteins with respect to horse cyt c led to several important conclusions. One of them is that Leu94 is always conserved in all 30 mammalian cyts c. It is known that mutation L94G of the wild type (WT) horse cyt c is destabilizing and mutant exists as molten globule under the native condition (buffer pH 6 and 25 °C). We have expressed and purified uniformly labeled (13C and 15N) and unlabeled WT horse cyt c and its L94G mutant. We report that labeling does not affect the thermodynamic stability of proteins. To support this conclusion, the secondary and tertiary structure of each protein in labeled and unlabeled forms was determined by conventional techniques (UV-Vis absorption and circular dichroism spectroscopy).
Asunto(s)
Citocromos c/metabolismo , Animales , Isótopos de Carbono/química , Dicroismo Circular , Citocromos c/química , Citocromos c/genética , Caballos , Concentración de Iones de Hidrógeno , Marcaje Isotópico , Mutagénesis Sitio-Dirigida , Isótopos de Nitrógeno/química , Pliegue de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
Position-specific isotope analysis by Nuclear Magnetic Resonance spectrometry was employed to study the 13C intramolecular isotopic fractionation associated with the migration of organic substrates through different stationary phases chromatography columns. Liquid chromatography is often used to isolate compounds prior to their isotope analysis and this purification step potentially alters the isotopic composition of target compounds introducing a bias in the later measured data. Moreover, results from liquid chromatography can yield the sorption parameters needed in reactive transport models that predict the transport and fate of organic contaminants to in the environment. The aim of this study was to use intramolecular isotope analysis to study both 13C and 15N isotope effects associated with the elution of paracetamol (acetaminophen) through different stationary phases and to compare them to effects observed previously for vanillin. Results showed very different intramolecular isotope fractionation profiles depending on the chemical structure of the stationary phase. The data also demonstrate that both the amplitude and the distribution of measured isotope effects depend on the nature of the non-covalent interactions involved in the migration process. Results provided by theoretical calculation performed during this study also confirmed the direct link between observed intramolecular isotope fractionation and the nature of involved intermolecular interactions. It is concluded that the nature of the stationary phase through which the substrate passes has a major impact on the intramolecular isotopic composition of organic compounds isolated by chromatography methods..