RESUMO
The 2C protein, which is an essential ATPase and one of the most conserved proteins across the Picornaviridae family, is an emerging antiviral target for which structural and functional characterization remain elusive. Based on a distant relationship to helicases of small DNA viruses, piconavirus 2C proteins have been predicted to unwind double-stranded RNAs. Here, a terminally extended variant of the 2C protein from echovirus 30 has been studied by means of enzymatic activity assays, transmission electron microscopy, atomic force microscopy and dynamic light scattering. The transmission electron-microscopy technique showed the existence of ring-shaped particles with â¼12 nm external diameter. Image analysis revealed that these particles were hexameric and resembled those formed by superfamily 3 DNA virus helicases.
Assuntos
Vírus de DNA/fisiologia , Enterovirus Humano B/fisiologia , RNA Helicases/química , Proteínas Recombinantes/química , Proteínas Virais/química , Vírion/química , Técnicas In Vitro , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Conformação Proteica , Multimerização Proteica , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Helicases/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Homologia Estrutural de Proteína , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/ultraestrutura , Vírion/ultraestruturaRESUMO
A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.