RESUMEN
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.
Asunto(s)
Virus ADN/fisiología , Enterovirus Humano B/fisiología , ARN Helicasas/química , Proteínas Recombinantes/química , Proteínas Virales/química , Virión/química , Técnicas In Vitro , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Conformación Proteica , Multimerización de Proteína , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Helicasas/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Homología Estructural de Proteína , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Virales/ultraestructura , Virión/ultraestructuraRESUMEN
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.