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La unión del ADN sensible a los iones de calcio en una proteína de fusión de un dedo de zinc
Akira Onoda1, Nozomi Arai, Naoto Shimazu
1Department of Chemistry, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo, 162-8601, Japan.
Journal of the American Chemical Society
|November 25, 2005
Resumen
Los investigadores diseñaron proteínas de dedo de zinc sensibles al calcio para el control de la unión al ADN. Estas nuevas proteínas de fusión demuestran una afinidad sintonizable de unión al ADN modulada por las concentraciones de iones de calcio, allanando el camino para los factores avanzados de transcripción artificial.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Biología Molecular Biología Molecular
- Ingeniería de proteínas Ingeniería de proteínas.
Sus antecedentes:
- Las proteínas del dedo de zinc son cruciales para el reconocimiento del ADN y la regulación génica.
- El desarrollo de proteínas de unión al ADN controlables es esencial para la biología sintética y la terapia génica.
- Los iones de calcio se pueden utilizar como un interruptor biológico para modular la función de las proteínas.
Objetivo del estudio:
- Diseñar nuevas proteínas de fusión de dedo de zinc con capacidades de unión de ADN sensibles al calcio.
- Para investigar la integridad estructural y el plegamiento de estas proteínas de fusión.
- Para evaluar la modulación dependiente de Ca de la afinidad de unión al ADN.
Principales métodos:
- Espectroscopia UV-vis y experimentos de dicroísmo circular (CD) para el análisis estructural.
- Espectroscopia de Resonancia Magnética Nuclear (RMN) y cálculos estructurales para estudios detallados de plegado.
- El ensayo de desplazamiento de movilidad electroforética (EMSA) para cuantificar la afinidad de unión al ADN bajo diferentes concentraciones de Ca2+.
Principales resultados:
- La proteína de fusión F2-Tn exhibió plegamiento nativo del dedo de zinc con un dominio integrado de unión de Ca.
- La proteína fusionada con troponina de dos dedos (F1F2-Tn) mostró unión al ADN Ca-responsive.
- F1F2-Tn mostró una constante de disociación (Kd) de 5,8 nM sin Ca2+ y 13 nM con 100 equivalentes de Ca2+.
Conclusiones:
- Las proteínas de dedo de zinc de fusión diseñadas artificialmente que incorporan un dominio de unión a la Ca exhiben una unión al ADN sensible a la Ca.
- Este trabajo proporciona una base para construir factores de transcripción artificiales basados en dedos de zinc con un interruptor de calcio.
- Las proteínas diseñadas ofrecen un nuevo mecanismo para una regulación genética precisa y controlada externamente.
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