RESUMO
The mitochondrial tyrosyl-tRNA synthetases (mt TyrRSs) of Pezizomycotina fungi are bifunctional proteins that aminoacylate mitochondrial tRNA(Tyr) and are structure-stabilizing splicing cofactors for group I introns. Studies with the Neurospora crassa synthetase (CYT-18 protein) showed that splicing activity is dependent upon Pezizomycotina-specific structural adaptations that form a distinct group I intron-binding site in the N-terminal catalytic domain. Although CYT-18's C-terminal domain also binds group I introns, it has been intractable to X-ray crystallography in the full-length protein. Here, we determined an NMR structure of the isolated C-terminal domain of the Aspergillus nidulans mt TyrRS, which is closely related to but smaller than CYT-18's. The structure shows an S4 fold like that of bacterial TyrRSs, but with novel features, including three Pezizomycontia-specific insertions. (15)N-(1)H two-dimensional NMR showed that C-terminal domains of the full-length A. nidulans and Geobacillus stearothermophilus synthetases do not tumble independently in solution, suggesting restricted orientations. Modeling onto a CYT-18/group I intron cocrystal structure indicates that the C-terminal domains of both subunits of the homodimeric protein bind different ends of the intron RNA, with one C-terminal domain having to undergo a large shift on its flexible linker to bind tRNA(Tyr) or the intron RNA on either side of the catalytic domain. The modeling suggests that the C-terminal domain acts together with the N-terminal domain to clamp parts of the intron's catalytic core, that at least one C-terminal domain insertion functions in group I intron binding, and that some C-terminal domain regions bind both tRNA(Tyr) and group I intron RNAs.
Assuntos
Processamento Alternativo , Íntrons , Espectroscopia de Ressonância Magnética/métodos , Tirosina-tRNA Ligase/química , Aspergillus nidulans/metabolismo , Domínio Catalítico , Cristalografia por Raios X/métodos , DNA/química , Geobacillus stearothermophilus/metabolismo , Modelos Moleculares , Modelos Teóricos , Conformação Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Solventes/químicaRESUMO
G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.
Assuntos
Detergentes/química , Escherichia coli/metabolismo , Engenharia de Proteínas , Receptores Acoplados a Proteínas G/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteases Dependentes de ATP/biossíntese , Proteases Dependentes de ATP/genética , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde/biossíntese , Humanos , Receptor B2 da Bradicinina/biossíntese , Receptor B2 da Bradicinina/química , Receptor B2 da Bradicinina/isolamento & purificação , Receptor CB1 de Canabinoide/biossíntese , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/isolamento & purificação , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , SolubilidadeRESUMO
A family of nanoparticles featuring surfaces of varying hydrophobicity was synthesized. The efficiency of DNA-binding was determined, demonstrating in a fivefold modulation in binding a 37-mer DNA strand. Nanoparticle-binding causes a reversible conformational change in the DNA structure, as demonstrated by circular dichroism and fluorescence experiments. Furthermore, the affinity of the nanoparticle for the DNA can be regulated by external agents, though stability of the complex is observed at relatively high ionic strengths.
Assuntos
DNA/metabolismo , Ouro/química , Nanoestruturas/química , Dicroísmo Circular , DNA/química , Ouro/metabolismo , Modelos Biológicos , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
Positively charged trimethylammonium-functionalized mixed monolayer protected clusters (MMPCs) bind DNA through complementary electrostatic interactions, resulting in complete inhibition of DNA transcription of T7 RNA polymerase. DNA was released from the nanoparticle by intracellular concentrations of glutathione, resulting in efficient transcription. The restoration of RNA production was dose-dependent in terms of GSH, with considerable control of the release process possible through variation in monolayer structure. This work presents a new approach to controlled release of DNA, with potential applications in the creation of transfection vectors and gene regulation systems.