RESUMEN
Metabolite profiling by NMR of body fluids is increasingly used to successfully differentiate patients from healthy individuals. Metabolites and their concentrations are direct reporters of body biochemistry. However, in blood plasma the NMR-detected free-metabolite concentrations are also strongly affected by interactions with the abundant plasma proteins, which have as of yet not been considered much in metabolic profiling. We previously reported that many of the common NMR-detected metabolites in blood plasma bind to human serum albumin (HSA) and many are released by fatty acids present in fatted HSA. HSA is the most abundant plasma protein and main transporter of endogenous and exogenous metabolites. Here, we show by NMR how the two most common fatty acids (FAs) in blood plasma - the long-chain FA, stearate (C18:0) and medium-chain FA, myristate (C14:0) - affect metabolite-HSA interaction. Of the set of 18 common NMR-detected metabolites, many are released by stearate and/or myristate, lactate appearing the most strongly affected. Myristate, but not stearate, reduces HSA-binding of phenylalanine and pyruvate. Citrate signals were NMR invisible in the presence of HSA. Only at high myristate-HSA mole ratios 11:1, is citrate sufficiently released to be detected. Finally, we find that limited dilution of blood-plasma mimics releases HSA-bound metabolites, a finding confirmed in real blood plasma samples. Based on these findings, we provide recommendations for NMR experiments for quantitative metabolite profiling.
Asunto(s)
Ácidos Grasos/análisis , Metabolómica/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Plasma/química , Albúmina Sérica/química , Aminoácidos/sangre , Citratos/sangre , Ácidos Grasos/metabolismo , Humanos , Lactatos/sangre , Modelos Moleculares , Ácido Mirístico/sangre , Fenilalanina/sangre , Ácido Pirúvico/sangre , Albúmina Sérica/metabolismo , Estearatos/sangreRESUMEN
The recently revised European Pharmacopeia and US Pharmacopeia heparin sodium monographs include nuclear magnetic resonance (NMR) tests on both identity and purity. In KMnO(4)-bleached heparin, an unidentified NMR signal is present at 2.10 ppm at a level of 15-20% of the mean of signal height of the major glucosamine (GlcNAc/GlcNS,6S) anomeric proton signal at 5.42 ppm and of the major iduronic acid (IdoA2S) anomeric proton signal at 5.21 ppm. According to the new monographs, no unidentified signals greater than 4% should be detected at that position. Thus, the material did not meet the acceptance criterion. The signal at 2.10 ppm has been present at the same level in all released MSD KMnO(4)-bleached heparin sodium batches analyzed over the past 10 years. The signal is a result of the KMnO(4) bleaching. No (oversulfated) chondroitin sulfate or dermatan sulfate was detected in this material. A comprehensive NMR study using long-range heteronuclear 2D techniques identifies this signal at 2.10 ppm as originating from the acetyl methyl group of (6-sulfated) 2-N-acetyl-2-deoxy-glucono-1,5-lactone. This modified monosaccharide is formed by the KMnO(4) oxidation of the reducing end of a terminal N-acetylglucosamine.
Asunto(s)
Anticoagulantes/química , Contaminación de Medicamentos , Heparina/química , Espectroscopía de Resonancia Magnética/métodos , Permanganato de Potasio/química , Sulfatos de Condroitina/análisis , Dermatán Sulfato/análisisRESUMEN
RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gag and pol open reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49 degrees, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.
Asunto(s)
Sistema de Lectura Ribosómico/genética , Regulación Viral de la Expresión Génica , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/metabolismo , Retrovirus de los Simios/genética , Adenina/metabolismo , Emparejamiento Base , Secuencia de Bases , Genes Virales/genética , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Resonancia Magnética Nuclear Biomolecular , Estabilidad del ARN/genética , ARN Viral/genética , TermodinámicaRESUMEN
The cleavage site of the Neurospora VS RNA ribozyme is located in a separate hairpin domain containing a hexanucleotide internal loop with an A-C mismatch and two adjacent G-A mismatches. The solution structure of the internal loop and helix la of the ribozyme substrate hairpin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The 2 nt in the internal loop, flanking the cleavage site, a guanine and adenine, are involved in two sheared G.A base pairs similar to the magnesium ion-binding site of the hammerhead ribozyme. Adjacent to the tandem G.A base pairs, the adenine and cytidine, which are important for cleavage, form a noncanonical wobble A+-C base pair. The dynamic properties of the internal loop and details of the high-resolution structure support the view that the hairpin structure represents a ground state, which has to undergo a conformational change prior to cleavage. Results of chemical modification and mutagenesis data of the Neurospora VS RNA ribozyme can be explained in context with the present three-dimensional structure.
Asunto(s)
Neurospora crassa/química , Conformación de Ácido Nucleico , ARN Catalítico/química , ARN de Hongos/química , Satélite de ARN/química , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Hidrólisis , Magnesio/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Neurospora crassa/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Recently, several high-resolution structures of-RNA pseudoknots have become available. Here we review the progress in this area. The majority of the structures obtained belong to the classical or H-type pseudoknot family. The most complicated pseudoknot structure elucidated so far is the Hepatitis Delta Virus ribozyme, which forms a nested double pseudoknot. In particular, the structure-function relationships of the H-type pseudoknots involved in translational frameshifting have received much attention. All molecules considered show interesting new structural motifs.
Asunto(s)
Conformación de Ácido Nucleico , ARN/química , Secuencia de Bases , Virus de la Hepatitis Delta/genética , Datos de Secuencia Molecular , ARN Catalítico/química , Relación Estructura-ActividadRESUMEN
The secondary structures of the 5'-untranslated region (5'-UTR) of five different tymoviruses have been determined by structure probing, computer prediction and sequence comparison. Despite large sequence differences, there are remarkable similarities in the secondary structure. In all viruses two or four hairpins are found, most of which contain a symmetrical internal loop consisting of adjacent C-C or C-A mismatches. Since it is known that such mismatches can be protonated and protonated cytosines play an important role in RNA-protein interactions in tymoviral virions, the influence of pH on the conformation of the internal loop was studied. UV melting experiments and 1-dimensional proton NMR at varying pH values and salt concentrations confirm that the hairpins can be protonated under relatively mild conditions. The hairpin found in the 5'-UTR of erysimum latent virus, which has an asymmetrical internal loop consisting of cytosines and uridines, shows comparable behaviour. It is concluded that all tymoviral RNAs contain protonatable hairpins in the 5'-UTR. Binding experiments with empty viral capsids, however, do not yet establish a role in capsid protein binding.