RESUMEN
This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless (13)C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Proteómica/métodos , Algoritmos , Interpretación Estadística de Datos , Modelos Moleculares , Proteínas/químicaRESUMEN
Four children, three girls aged 9, 5.5 and 6.5 years, and a boy aged 8 years, suffered from urinary incontinence. The underlying conditions, urge syndrome with urge incontinence, ectopic ureter, meatus stenosis, and pelvic floor dysfunction, respectively, were identified and treated, after which the children were no longer incontinent. Diurnal incontinence is present in 2-3% of 7-year-olds. Incontinence is accompanied by recurrent urinary tract infections and vesicoureteric reflux in 40% of cases. A combination of these two disorders can lead to impaired renal function. Especially in girls, involuntary urine loss is usually based on dysfunctional voiding, a non-neurogenic coordination problem between the detrusor muscle of the bladder and the pelvic floor. A careful history is the most important tool in reaching the correct diagnosis.
Asunto(s)
Incontinencia Urinaria/diagnóstico , Infecciones Urinarias/complicaciones , Reflujo Vesicoureteral/complicaciones , Niño , Diagnóstico Diferencial , Femenino , Humanos , Riñón/fisiopatología , Masculino , Recurrencia , Incontinencia Urinaria/complicaciones , Incontinencia Urinaria/terapiaRESUMEN
The determination by NMR of the solution structure of the phosphorylated enzyme IIB (P-IIB(Chb)) of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli is presented. Most of the backbone and side-chain resonances were assigned using a variety of mostly heteronuclear NMR experiments. The remaining resonances were assigned with the help of the structure calculations.NOE-derived distance restraints were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In addition, combinations of ambiguous restraints were used to resolve ambiguities in the NOE assignments. By combining sets of ambiguous and unambiguous restraints into new ambiguous restraints, an error function was constructed that was less sensitive to information loss caused by assignment uncertainties. The final set of structures had a pairwise rmsd of 0.59 A and 1.16 A for the heavy atoms of the backbone and side-chains, respectively. Comparing the P-IIB(Chb) solution structure with the previously determined NMR and X-ray structures of the wild-type and the Cys10Ser mutant shows that significant differences between the structures are limited to the active-site region. The phosphoryl group at the active-site cysteine residue is surrounded by a loop formed by residues 10 through 16. NOE and chemical shift data suggest that the phosphoryl group makes hydrogen bonds with the backbone amide protons of residues 12 and 15. The binding mode of the phosphoryl group is very similar to that of the protein tyrosine phosphatases. The differences observed are in accordance with the presumption that IIB(Chb) has to be more resistant to hydrolysis than the protein tyrosine phosphatases. We propose a proton relay network by which a transfer occurs between the cysteine SH proton and the solvent via the hydroxyl group of Thr16.
Asunto(s)
Cisteína/metabolismo , Disacáridos/metabolismo , Escherichia coli/enzimología , Resonancia Magnética Nuclear Biomolecular , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Mutación , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Fosforilación , Estructura Secundaria de Proteína , Protones , Solventes , Especificidad por Sustrato , TermodinámicaRESUMEN
Three patients, two boys of 5 months and 6 years and one girl aged 4 years, presented with acute abdominal pain, vomiting and fever, suggesting peritonitis. Imaging examinations (abdominal survey roentgenogram and (or) echography), exploratory laparotomy (in two patients) and blood cultures with growth of Streptococcus pneumoniae led to the diagnosis of primary peritonitis. Intravenous antibiotics led to recovery, in one patient complicated by paralytic ileus, which was treated surgically. Primary peritonitis is a rare condition which should be considered in the differential diagnosis of children with an acute abdominal syndrome. Conditions requiring surgery should be excluded by imaging examinations or laparotomy. When the diagnosis is confirmed by paracentesis or laparotomy, antibiotic treatment has to be started.
Asunto(s)
Peritonitis/diagnóstico , Streptococcus pneumoniae/aislamiento & purificación , Dolor Abdominal/etiología , Niño , Preescolar , Femenino , Fiebre/etiología , Humanos , Masculino , Peritonitis/microbiología , Vómitos/etiologíaRESUMEN
The assignment of the side-chain NMR resonances and the determination of the three-dimensional solution structure of the C10S mutant of enzyme IIBcellobiose (IIBcel) of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli are presented. The side-chain resonances were assigned nearly completely using a variety of mostly heteronuclear NMR experiments, including HCCH-TOCSY, HCCH-COSY, and COCCH-TOCSY experiments as well as CBCACOHA, CBCA(CO)NH, and HBHA(CBCA)(CO)NH experiments. In order to obtain the three-dimensional structure, NOE data were collected from 15N-NOESY-HSQC, 13C-HSQC-NOESY, and 2D NOE experiments. The distance restraints derived from these NOE data were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In an iterative procedure, additional NOE assignments were derived from the calculated structures and new structures were calculated. The final set of structures, calculated with approximately 2000 unambiguous and ambiguous distance restraints, has an rms deviation of 1.1 A on C alpha atoms. IIBcel consists of a four stranded parallel beta-sheet, in the order 2134. The sheet is flanked with two and three alpha-helices on either side. Residue 10, a cysteine in the wild-type enzyme, which is phosphorylated during the catalytic cycle, is located at the end of the first beta-strand. A loop that is proposed to be involved in the binding of the phosphoryl-group follows the cysteine. The loop appears to be disordered in the unphosphorylated state.
Asunto(s)
Escherichia coli/enzimología , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Secuencia de Aminoácidos , Isótopos de Carbono , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Protones , SolucionesRESUMEN
The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone 1H, 15N, and 13C alpha resonances, and the HN(CA)CO and HNCO experiments, correlating backbone 1H,15N and 13CO resonances. Heteronuclear 1H-NOE 1H-15N single quantum coherence (15N-NOESY-HSQC) spectroscopy and heteronuclear 1H total correlation 1H-15N single quantum coherence (15N-TOCSY-HSQC) spectroscopy were used to resolve ambiguities arising from overlapping 13C alpha and 13CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3-dimensional 1H alpha-13C alpha-13CO experiment (COCAH), yielded the assignment for all observed backbone resonances. The secondary structure was determined using information both from the deviation of observed 1H alpha and 13C alpha chemical shifts from their random coil values and 1H-NOE information from the 15N-NOESY-HSQC. These data show that enzyme IIBcellobiose consists of a 4-stranded parallel beta-sheet and 5 alpha-helices. In the wild-type enzyme IIBcellobiose, the catalytic residue appears to be located at the end of a beta-strand.
Asunto(s)
Escherichia coli/enzimología , Espectroscopía de Resonancia Magnética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , Mutagénesis , Estructura Secundaria de ProteínaRESUMEN
Phospholipases A2 play a part in a number of physiologically important cellular processes such as inflammation, blood platelet aggregation and acute hypersensitivity. These processes are all initiated by the release of arachidonic acid from cell membranes which is catalysed by intracellular phospholipases A2 and followed by conversion of arachidonic acid to prostaglandins, leukotrienes or thromboxanes. An imbalance in the production of these compounds can lead to chronic inflammatory diseases such as rheumatoid arthritis and asthma. Inhibitors of phospholipase A2 might therefore act to reduce the effects of inflammation, so structural information about the binding of phospholipase A2 to its substrates could be helpful in the design of therapeutic drugs. The three-dimensional structure is not known for any intracellular phospholipase A2, but these enzymes share significant sequence homology with secreted phospholipases, for which some of the structures have been determined. Here we report the structure of a complex between an extracellular phospholipase A2 and a competitively inhibiting substrate analogue, which reveals considerable detail about the interaction and suggests a mechanism for catalysis by this enzyme.