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Síntesis de complejos de proteínas múltiples mediante activación secuencial dirigida por carga de residuos de

Casey S Mogilevsky¹, Marco J Lobba¹, Daniel D Brauer¹

¹Department of Chemistry, University of California, Berkeley, California 94720, United States.

Journal of the American Chemical Society

|August 12, 2021

Resumen

Los investigadores diseñaron una nueva enzima tirosinasa (megaTYR) para el acoplamiento selectivo de proteínas. Esta enzima permite modificaciones precisas de las proteínas, facilitando la construcción de estructuras complejas de proteínas como los trímeres.

Área de la Ciencia:

Biología Química
Enzimología
Ingeniería de proteínas

Sus antecedentes:

El acoplamiento proteico-proteico selectivo del sitio es un desafío clave en biología química.
Las enzimas tirosinasa pueden convertir los residuos de tirosina en ortoquinonas reactivas para el acoplamiento.
Las enzimas anteriores como la tirosinasa de Agaricus bisporus (abTYR) tienen limitaciones en la producción y el alcance del sustrato.

Objetivo del estudio:

Identificar y diseñar una nueva tirosinasa con mejor tolerancia al sustrato para el acoplamiento de proteínas.
Desarrollar un nuevo método para la modificación y el ensamblaje selectivo de proteínas.
Demostrar la utilidad de la tirosinasa diseñada en la construcción de arquitecturas complejas de proteínas.

Principales métodos:

Selección de varios candidatos a la tirosinasa.
Ingeniería de proteínas de Bacillus megaterium tirosinasa (megaTYR) a través de mutaciones en un solo punto.
Desarrollo de una estrategia de activación secuencial de residuos de tirosina dirigida por carga (CDSAT).
Construcción y caracterización de los recortadores de proteínas.

Principales resultados:

El Bacillus megaterium tyrosinase (megaTYR) tiene una amplia tolerancia al sustrato.
Las variantes de megaTYR diseñadas pueden activar residuos de tirosina en diversos contextos de secuencia.
El método de activación secuencial dirigida por carga de los residuos de tirosina (CDSAT) permitió con éxito la construcción de trimadores de proteínas.

Conclusiones:

El megaTYR de ingeniería es una herramienta versátil para la modificación de proteínas selectivas del sitio.
La estrategia CDSAT proporciona un nuevo enfoque para el ensamblaje de estructuras de proteínas.
Este trabajo amplía el conjunto de herramientas para la ingeniería de proteínas y la bioconjugación.

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