RESUMEN
An analog of pyrazinamide (PZA), 5-chloropyrazinamide (5-Cl-PZA), has previously been shown to inhibit mycobacterial fatty acid synthase I (FASI). FASI has been purified from a recombinant strain of M. smegmatis (M. smegmatis Deltafas1 attB::M. tuberculosis fas1). Following purification, FASI activity and inhibition were assessed spectrophotometrically by monitoring NADPH oxidation. The observed inhibition was both concentration and structure dependent, being affected by both substitution at the 5 position of the pyrazine nucleus and the nature of the ester or N-alkyl group. Under the conditions studied, both 5-Cl-PZA and PZA exhibited concentration and substrate dependence consistent with competitive inhibition of FASI with K(i)s of 55 to 59 microM and 2,567 to 2,627 microM, respectively. The results were validated utilizing a radiolabeled fatty acid synthesis assay. This assay showed that FASI was inhibited by PZA and pyrazinoic acid as well as by a series of PZA analogs.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Ácido Graso Sintasas/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Pirazinamida/análogos & derivados , Pirazinamida/farmacología , Proteínas Bacterianas/aislamiento & purificación , Sistema Libre de Células , Ácido Graso Sintasas/aislamiento & purificación , Humanos , Concentración 50 Inhibidora , Cinética , Espectrometría de Masas , Estructura Molecular , NADP/metabolismo , Oxidación-Reducción , Pirazinamida/metabolismo , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
The design and rapid construction of libraries of genes coding beta-sheet forming repetitive and block-copolymerized polypeptides bearing various C- and N-terminal sequences are described. The design was based on the assembly of DNA cassettes coding for the (GA)3GX amino acid sequence where the (GAGAGA) sequences would constitute the beta-strand units of a larger beta-sheet assembly. The edges of this beta-sheet would be functionalized by the turn-inducing amino acids (GX). The polypeptides were expressed in Escherichia coli using conventional vectors and were purified by Ni-nitriloacetic acid (NTA) chromatography. The correlation of polymer structure with molecular weight was investigated by gel electrophoresis and mass spectrometry. The monomer sequences and post-translational chemical modifications were found to influence the mobility of the polypeptides over the full range of polypeptide molecular weights while the electrophoretic mobility of lower molecular weight polypeptides was more susceptible to C- and N-termini polypeptide modifications.
Asunto(s)
Biblioteca de Péptidos , Péptidos/química , Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Cromatografía , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Biblioteca de Genes , Datos de Secuencia Molecular , Peso Molecular , Ácido Nitrilotriacético/análogos & derivados , Ácido Nitrilotriacético/química , Compuestos Organometálicos/química , Péptidos/genética , Estructura Secundaria de Proteína , Secuencias Repetitivas de AminoácidoRESUMEN
A de novo, genetically engineered 687 residue polypeptide expressed in E. coli has been found to form highly rectilinear, beta-sheet containing fibrillar structures. Tapping-mode atomic force microscopy, deep-UV Raman spectroscopy, and transmission electron microscopy definitively established the tendency of the fibrils to predominantly display an apparently planar bilayer or ribbon assemblage. The ordered self-assembly of designed, extremely repetitive, high molecular weight peptides is a harbinger of the utility of similar materials in nanoscience and engineering applications.
Asunto(s)
Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/síntesis química , Péptidos/química , Péptidos/síntesis química , Ingeniería de Proteínas , Secuencia de Bases , ADN/química , ADN/genética , Membrana Dobles de Lípidos/aislamiento & purificación , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Peso Molecular , Tamaño de la Partícula , Péptidos/aislamiento & purificación , Conformación Proteica , Estructura Secundaria de Proteína , Sensibilidad y Especificidad , Espectrometría RamanRESUMEN
A variety of mono- and difluoroacetylsilanes and the corresponding silyl enol ethers were prepared from trifluoroethanol and chlorotrialkylsilanes in the presence of LDA through retro-Brook rearrangement. Sterically demanding silyl groups, especially those bound to oxygen, resulted in higher yields of difluoroacetylsilanes. The yields of difluoroacetylsilanes were also dramatically affected by the method of the termination of the reaction. Difluorohaloacetylsilanes were prepared from the corresponding difluoroethenyl silyl ethers with electrophilic halogenating reagents in good yields. A gamma-fluorinated beta-diketone 9a was prepared from monofluoroacetyltriisopropylsilane by nucleophilic acylation with methyl trifluoroacetate.
