López A, Valle A, Pedrera L, Martínez D, Fando R, Schreier S, Álvarez C, Lanio ME, Pazos F

Idioma: Ingles.
Referencias bibliográficas: 38
Paginas: 13-18
Archivo PDF: 211.76 Kb.

RESUMEN

El mecanismo de formación de poros de las actinoporinas es un proceso de varias etapas hasta la formación de un poro funcional. Se requiere un sitio de unión a fosfocolina, un grupo de anillos aromáticos y una región básica, para la interacción inicial con la membrana. La región N-terminal es relevante para la formación del poro. En este trabajo se diseñó y obtuvo dos mutantes de Sticolisina I (St I) con Cys en regiones funcionalmente relevantes para la interacción con membranas: St I E2C (en la región N-terminal) y St I R52C (en el sitio de unión a membranas). El reemplazo de los residuos Glu-2 y Arg-52 por Cys no produce cambios notables en la conformación de St I, según determinaciones de fluorescencia y espectroscopía de dicroísmo circular. El primer cambio no afectó la actividad permeabilizante. La disminución relativa en la capacidad formadora de poros en St I R52C no se vincula a su menor capacidad de asociación con la membrana. St I E2C y St I R52C conservan las principales características conformacionales de St I nativa y muestran similar capacidad de unión a vesículas liposomales, mientras que difieren en cuanto a actividad permeabilizante. Estos dos mutantes son herramientas útiles para estudiar los pasos de los mecanismos de permeabilización de las Sticholisinas, en especial los que ocurren tras su unión a las membranas, mediante el uso de sondas específicas para grupos tiol tales como indicadores fluorescentes y del momento del espín.

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