RESUMO
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.
Assuntos
Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Cromatografia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Biblioteca Gênica , Dados de Sequência Molecular , Peso Molecular , Ácido Nitrilotriacético/análogos & derivados , Ácido Nitrilotriacético/química , Compostos Organometálicos/química , Peptídeos/genética , Estrutura Secundária de Proteína , Sequências Repetitivas de AminoácidosRESUMO
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.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Ácido Graxo Sintases/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Pirazinamida/análogos & derivados , Pirazinamida/farmacologia , Proteínas de Bactérias/isolamento & purificação , Sistema Livre de Células , Ácido Graxo Sintases/isolamento & purificação , Humanos , Concentração Inibidora 50 , Cinética , Espectrometria de Massas , Estrutura Molecular , NADP/metabolismo , Oxirredução , Pirazinamida/metabolismo , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
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.
Assuntos
Bicamadas Lipídicas/química , Bicamadas Lipídicas/síntese química , Peptídeos/química , Peptídeos/síntese química , Engenharia de Proteínas , Sequência de Bases , DNA/química , DNA/genética , Bicamadas Lipídicas/isolamento & purificação , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Peso Molecular , Tamanho da Partícula , Peptídeos/isolamento & purificação , Conformação Proteica , Estrutura Secundária de Proteína , Sensibilidade e Especificidade , Análise Espectral RamanRESUMO
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.