RESUMO
The A19 protein of vaccinia virus (VACV) is conserved among chordopoxviruses, expressed late in infection, packaged in the virus core, and required for a late step in morphogenesis. Multiple-sequence alignments of A19 homologs indicated conservation of a series of lysines and arginines, which could represent a nuclear localization or nucleic acid binding motif, and a pair of CXXC motifs that suggested a zinc finger or redox active sites. The importance of the CXXC motif was confirmed by cysteine-to-serine substitutions, which rendered the altered protein unable to trans-complement infectivity of a null mutant. Nevertheless, the cysteines were not required for function of the poxvirus-specific redox pathway. Epitope-tagged A19 proteins were detected in the nucleus and cytoplasm in both infected and uninfected cells, but this distribution was unaffected by alanine substitutions of the arginine residues, which only partially reduced the ability of the mutated protein to trans-complement infectivity. Viral proteins specifically associated with affinity-purified A19 were identified by mass spectrometry as components of the transcription complex, including RNA polymerase subunits, RAP94 (RNA polymerase-associated protein 94), early transcription factors, capping enzyme, and nucleoside triphosphate phosphohydrolase I, and two core proteins required for morphogenesis. Further studies suggested that the interaction of A19 with the RNA polymerase did not require RAP94 or other intermediate or late viral proteins but was reduced by mutation of cysteines in the putative zinc finger domain. Although A19 was not required for incorporation of the transcription complex in virus particles, the transcriptional activity of A19-deficient virus particles was severely reduced.
Assuntos
Vaccinia virus/patogenicidade , Vacínia/virologia , Proteínas Virais/metabolismo , Vírion/patogenicidade , Montagem de Vírus , Sequência de Aminoácidos , Animais , Western Blotting , Chlorocebus aethiops , Cromatografia de Afinidade , Citoplasma/metabolismo , Citoplasma/virologia , Replicação do DNA , DNA Viral/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Células HeLa , Humanos , Rim/citologia , Rim/metabolismo , Rim/virologia , Dados de Sequência Molecular , Mutação/genética , Fases de Leitura Aberta , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vacínia/genética , Vacínia/metabolismo , Proteínas Virais/genética , Replicação ViralRESUMO
Efforts to develop a vaccine for the elimination of malaria include the use of carrier proteins to assemble monomeric antigens into nanoparticles to maximize immunogenicity. Recombinant ExoProtein A (EPA) is a detoxified form of Pseudomonas aeruginosa Exotoxin A which has been used as a carrier in the conjugate vaccine field. A pilot-scale process developed for purification of EPA yielded product that consistently approached a preset upper limit for host cell protein (HCP) content per human dose. To minimize the risk of bulk material exceeding the specification, the purification process was redeveloped using mixed-mode chromatography resins. Purified EPA derived from the primary and redeveloped processes were comparable following full biochemical and biophysical characterization. However, using a process specific immunoassay, the HCP content was shown to decrease from a range of 0.14-0.24% w/w of total protein to below the level of detection with the revised process. The improved process reproducibly yields EPA with highly similar quality characteristics as the original process but with an improved profile for the HCP content.
Assuntos
ADP Ribose Transferases/química , ADP Ribose Transferases/imunologia , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Fenômenos Químicos , Exotoxinas/química , Exotoxinas/imunologia , Vacinas contra Pseudomonas/química , Vacinas contra Pseudomonas/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia , Fatores de Virulência/química , Fatores de Virulência/imunologia , ADP Ribose Transferases/isolamento & purificação , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/isolamento & purificação , Epitopos/imunologia , Exotoxinas/isolamento & purificação , Humanos , Imunogenicidade da Vacina , Camundongos , Peptídeos/imunologia , Processamento de Proteína Pós-Traducional , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Análise Espectral , Vacinas Sintéticas/isolamento & purificação , Fatores de Virulência/isolamento & purificação , Exotoxina A de Pseudomonas aeruginosaRESUMO
Carbapenem-resistant Klebsiella pneumoniae strains have emerged as a cause of life-threatening infections in susceptible individuals (e.g., transplant recipients and critically ill patients). Strains classified as multilocus sequence type (ST) 258 are among the most prominent causes of carbapenem-resistant K. pneumoniae infections worldwide, but the basis for the success of this lineage remains incompletely determined. To gain a more comprehensive view of the molecules potentially involved in the success of ST258, we used a proteomics approach to identify surface-associated and culture supernatant proteins produced by ST258. Protein samples were prepared from varied culture conditions in vitro, and were analyzed by a combination of two-dimensional electrophoresis and liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). We identified a total of 193 proteins in outer membrane preparations from bacteria cultured in Luria-Bertani broth (LB) or RPMI 1640 tissue culture media (RPMI). Compared with LB, several iron-acquisition proteins, including IutA, HmuR, HmuS, CirA, FepA, FitA, FoxA, FhuD, and YfeX, were more highly expressed in RPMI. Of the 177 proteins identified in spent media, only the fimbrial subunit, MrkA, was predicted to be extracellular, a finding that suggests few proteins (or a limited quantity) are freely secreted by ST258. Notably, we discovered 203 proteins not reported in previous K. pneumoniae proteome studies. In silico modeling of proteins with unknown function revealed several proteins with beta-barrel transmembrane structures typical of porins, as well as possible host-interacting proteins. Taken together, these findings contribute several new targets for the mechanistic study of drug-resistance and pathogenesis by ST258 K. pneumoniae isolates.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Técnicas de Tipagem Bacteriana , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Klebsiella pneumoniae/classificação , Klebsiella pneumoniae/metabolismo , Tipagem de Sequências Multilocus , Proteínas da Membrana Bacteriana Externa/química , Meios de Cultura , Eletroforese em Gel Bidimensional , Klebsiella pneumoniae/efeitos dos fármacos , Espectrometria de MassasRESUMO
A new method for performing molecular dynamics simulations with fluctuating charge polarizable potentials is introduced. In fluctuating charge models, polarizability is treated by allowing the partial charges to be variables, with values that are coupled to charges on the same molecule as well as those on other molecules. The charges can be efficiently propagated in a molecular dynamics simulation using extended Lagrangian dynamics. By making a coordinate change from the charge variables to a set of normal mode charge coordinates for each molecule, a new method is constructed in which the normal mode charge variables uncouple from those on the same molecule. The method is applied to the TIP4P-FQ model of water and compared to other methods for implementing the dynamics. The methods are compared using different molecular dynamics time steps.
RESUMO
Free energy calculations for the transfer of a water molecule from the pure liquid to an interior cavity site in a protein are presented. Two different protein cavities, in bovine pancreatic trypsin inhibitor (BPTI) and in the I76A mutant of barnase, represent very different environments for the water molecule: one which is polar, forming four water-protein hydrogen bonds, and one which is more hydrophobic, forming only one water-protein hydrogen bond. The calculations give very different free energies for the different cavities, with only the polar BPTI cavity predicted to be hydrated. The corresponding entropies for the transfer to the interior cavities are calculated as well and show that the transfer to the polar cavity is significantly entropically unfavorable while the transfer to the nonpolar cavity is entropically favorable. For both proteins an analysis of the fluctuations in the positions of the protein atoms shows that the addition of a water molecule makes the protein more flexible. This increased flexibility appears to be due to an increased length and weakened strength of protein-protein hydrogen bonds near the cavity.