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La ruptura de la simetría de carga-desproporción crea un complejo de mioglobina heterodímero con afinidad mejorada y
Ethan N Trana1, Judith M Nocek1, Jon Vander Woude1
1Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
Journal of the American Chemical Society
|September 21, 2016
Resumen
Diseñamos un dímero de mioglobina (Mb) con una afinidad de unión significativamente mejorada a través de la desproporción de carga. Este avance permite una rápida transferencia de electrones entre proteínas (ET) con constantes de velocidad aumentadas en más de cinco órdenes de magnitud.
Área de la Ciencia:
- La bioquímica
- Ingeniería de proteínas
- Dinámica de transferencia de electrones
Sus antecedentes:
- La mioglobina (Mb) funciona típicamente como un monómero.
- La ingeniería de dimeros Mb presenta desafíos para lograr complejos estables y transferencia de electrones controlada.
- Los estudios previos sobre la transferencia de electrones entre proteínas en dímeros Mb se enfrentaron a limitaciones debido a la entrada dinámica.
Objetivo del estudio:
- Diseñar un dímero de mioglobina (Mb) con una afinidad de unión muy mejorada y una transferencia intracompleja de electrones (ET) controlada.
- Investigar los efectos de la desproporción de la carga en la formación de dímeros Mb y la cinética ET.
- Desarrollar y validar nuevas estrategias de ingeniería de proteínas para crear complejos de proteínas funcionales.
Principales métodos:
- Simulaciones de dinámica browniana (BD) para el rediseño de la interfaz, incluido un nuevo enfoque de "filtro hemo".
- Mutagénesis dirigida al sitio para introducir cargas superficiales complementarias positivas (Mb(+6) y negativas (Mb(-6).
- Las mediciones del fotociclo de expresión, caracterización por rayos X y transferencia de electrones fotoiniciados (ET) utilizando Mb sustituido por Zn-deuteroporfirina.
Principales resultados:
- Se crearon con éxito mutantes complementarios Mb(+6) y Mb(-6) que forman un dímero estable con bordes casi paralelos a una distancia de ~ 18-19 Å.
- La desproporción de carga aumentó la constante de unión en al menos 5 órdenes de magnitud en comparación con el tipo salvaje Mb.
- Las constantes de velocidad ET intracomplejas para la separación de cargas (CS) y la recombinación de cargas (CR) aumentaron en más de 5 órdenes de magnitud, exhibiendo una independencia de viscosidad indicativa de una verdadera ET interproteína.
- Se observó una reaparición sin precedentes del intermedio de separación de carga I después de la desintegración inicial, lo que sugiere un comportamiento cinético complejo.
Conclusiones:
- Los dimeros Mb diseñados con desproporción de carga exhiben una afinidad de unión dramáticamente mejorada y una ET interproteína rápida.
- Las estrategias de simulación y mutagénesis de BD desarrolladas son efectivas para el diseño de interacciones proteína-proteína funcionales.
- La cinética ET observada y el comportamiento intermedio proporcionan nuevos conocimientos sobre los mecanismos de transferencia de electrones fotoiniciados en complejos de proteínas.
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