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1.
PLoS One ; 6(8): e14827, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21853016

RESUMEN

UNLABELLED: The parasite Cryptosporidium parvum has three 14-3-3 proteins: Cp14ε, Cp14a and Cp14b, with only Cp14ε similar to human 14-3-3 proteins in sequence, peptide-binding properties and structure. Structurally, Cp14a features the classical 14-3-3 dimer but with a uniquely wide pocket and a disoriented RRY triad potentially incapable of binding phosphopeptides. The Cp14b protein deviates from the norm significantly: (i) In one subunit, the phosphorylated C-terminal tail is bound in the binding groove like a phosphopeptide. This supports our binding study indicating this protein was stabilized by a peptide mimicking its last six residues. (ii) The other subunit has eight helices instead of nine, with αA and αB forming a single helix and occluding the peptide-binding cleft. (iii) The protein forms a degenerate dimer with the two binding grooves divided and facing opposite directions. These features conspire to block and disrupt the bicameral substrate-binding pocket, suggesting a possible tripartite auto-regulation mechanism that has not been observed previously. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.


Asunto(s)
Proteínas 14-3-3/metabolismo , Cryptosporidium parvum/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas 14-3-3/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Protozoarias/química , Alineación de Secuencia
2.
Biochemistry ; 44(30): 10339-48, 2005 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-16042411

RESUMEN

We report the biochemical and biophysical characterization of YedYZ, a sulfite oxidase homologue from Escherichia coli. YedY is a soluble catalytic subunit with a twin arginine leader sequence for export to the periplasm by the Tat translocation system. YedY is the only molybdoenzyme so far isolated from E. coli with the Mo-MPT form of the molybdenum cofactor. The electron paramagnetic resonance (EPR) signal of the YedY molybdenum is similar to that of known Mo-MPT containing enzymes, with the exception that only the Mo(IV) --> Mo(V) transition is observed, with a midpoint potential of 132 mV. YedZ is a membrane-intrinsic cytochrome b with six putative transmembrane helices. The single heme b of YedZ has a midpoint potential of -8 mV, determined by EPR spectroscopy of YedZ-enriched membrane preparations. YedY does not associate strongly with YedZ on the cytoplasmic membrane. However, mutation of the YedY active site Cys102 to Ser results in very efficient targeting of YedY to YedZ in the membrane, demonstrating a clear role for YedZ as the membrane anchor for YedY. Together, YedYZ comprise a well-conserved bacterial heme-molybdoenzyme found in a variety of bacteria that can be assigned to the sulfite oxidase class of enzyme.


Asunto(s)
Cisteína/química , Proteínas de Escherichia coli/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Procesamiento Proteico-Postraduccional , Sitios de Unión/genética , Biología Computacional , Secuencia Conservada/genética , Cisteína/genética , Espectroscopía de Resonancia por Spin del Electrón , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/fisiología , Histidina/química , Mutagénesis Sitio-Dirigida , Oligopéptidos/química , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/aislamiento & purificación , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/fisiología , Serina/genética , Homología Estructural de Proteína , Especificidad por Sustrato/genética , Tungsteno/química
3.
Mol Cell Proteomics ; 4(8): 1205-9, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15911532

RESUMEN

Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. Represented within this inventory were unique and modified forms corresponding to 575 different ORFs that included 151 proteins whose existence had been predicted from hypothetical ORFs, 76 proteins of completely unknown function, and 222 proteins currently without location assignments in the Swiss-Prot Database. Of the 575 unique proteins identified, 42% were found to exist in multiple forms. Using DIGE, we also examined the relative changes in protein expression when cells were grown in the presence and absence of amino acids. A total of 23 different proteins were identified whose abundance changed significantly between the two conditions. Most of these changes were found to be associated with proteins involved in carbon and amino acid metabolism, transport, and chemotaxis. Detailed information related to all 2,160 protein forms (protein and gene names, accession numbers, subcellular locations, relative abundances, sequence coverage, molecular masses, and isoelectric points) can be obtained upon request in either tabular form or as interactive gel images.


Asunto(s)
Aminoácidos/deficiencia , Proteínas Bacterianas/química , Escherichia coli/química , Escherichia coli/crecimiento & desarrollo , Genoma Bacteriano , Proteínas Bacterianas/aislamiento & purificación , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional/métodos , Sistemas de Lectura Abierta , Mapeo Peptídico/métodos , Análisis de Secuencia/métodos
4.
J Biol Chem ; 279(48): 50391-400, 2004 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-15355966

RESUMEN

By using a bioinformatics screen of the Escherichia coli genome for potential molybdenum-containing enzymes, we have identified a novel oxidoreductase conserved in the majority of Gram-negative bacteria. The identified operon encodes for a proposed heterodimer, YedYZ in Escherichia coli, consisting of a soluble catalytic subunit termed YedY, which is likely anchored to the membrane by a heme-containing trans-membrane subunit termed YedZ. YedY is uniquely characterized by the presence of one molybdenum molybdopterin not conjugated by an additional nucleotide, and it represents the only molybdoenzyme isolated from E. coli characterized by the presence of this cofactor form. We have further characterized the catalytic subunit YedY in both the molybdenum- and tungsten-substituted forms by using crystallographic analysis. YedY is very distinct in overall architecture from all known bacterial reductases but does show some similarity with the catalytic domain of the eukaryotic chicken liver sulfite oxidase. However, the strictly conserved residues involved in the metal coordination sphere and in the substrate binding pocket of YedY are strikingly different from that of chicken liver sulfite oxidase, suggesting a catalytic activity more in keeping with a reductase than that of a sulfite oxidase. Preliminary kinetic analysis of YedY with a variety of substrates supports our proposal that YedY and its many orthologues may represent a new type of membrane-associated bacterial reductase.


Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/enzimología , Oxidorreductasas/genética , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Datos de Secuencia Molecular , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Estructura Terciaria de Proteína , Alineación de Secuencia
5.
J Bacteriol ; 186(10): 3254-8, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15126489

RESUMEN

To gain insight into the cell envelope of Escherichia coli grown under aerobic and anaerobic conditions, lipoproteins were examined by using functional genomics. The mRNA expression levels of each of these genes under three growth conditions--aerobic, anaerobic, and anaerobic with nitrate--were examined by using both Affymetrix GeneChip E. coli antisense genome arrays and real-time PCR (RT-PCR). Many genes showed significant changes in expression level. The RT-PCR results were in very good agreement with the microarray data. The results of this study represent the first insights into the possible roles of unknown lipoprotein genes and broaden our understanding of the composition of the cell envelope under different environmental conditions. Additionally, these data serve as a test set for the refinement of high-throughput bioinformatic and global gene expression methods.


Asunto(s)
Escherichia coli/genética , Genoma Bacteriano , Lipoproteínas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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