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Asignación de resonancia específica de secuencia de proteínas no globulares solubles mediante espectroscopia 7D

Sebastian Hiller¹, Christian Wasmer, Gerhard Wider

¹Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.

Journal of the American Chemical Society

|August 19, 2007

Resumen

Este estudio introduce un método automatizado de RMN para el análisis rápido y de resolución atómica de proteínas desplegadas. Esta técnica acelera significativamente el estudio de las proteínas no globulares, permitiendo nuevos conocimientos sobre su estructura y función.

Área de la Ciencia:

La biofísica es la biofísica.
Biología Estructural Biología estructural.
Espectroscopia de Resonancia Magnética Nuclear (RMN) Espectroscopía de Resonancia Magnética Nuclear (RMN) Espectroscopía de Resonancia Magnética Nuclear (RMN) Espectroscopía de Resonancia Magnética Nuclear (RMN) Espectroscopía de Resonancia Magnética Nuclear (RMN) Espectroscopía de Resonancia Magnética Nuclear (RMN) Espectroscopía de Resonancia Magnética Nuclear (RMN) Espectroscopía de Resonancia Magnética Nuclear (RMN) Espectroscopía de Resonancia Magnética Nuclear (RMN) Espectroscopía de Resonancia Magnética Nuclear (RMN) Espectroscopía de Resonancia Magnética Nuclear (RMN)

Sus antecedentes:

La espectroscopia de RMN proporciona datos de resolución atómica para las proteínas no globulares solubles.
La asignación por RMN de polipéptidos desplegados es un desafío debido a la dispersión de desplazamiento químico limitado, lo que dificulta las aplicaciones prácticas.

Objetivo del estudio:

Desarrollar un método eficiente y totalmente automatizado para la asignación de secuencias específicas de RMN de proteínas desplegadas.
Para permitir estudios de resolución atómica de proteínas químicamente desnaturalizadas y polipéptidos no globulares funcionales.

Principales métodos:

Utilizó nuevos experimentos de espectroscopia de proyección automatizada (APSY).
Registran conjuntos de datos de RMN de cinco a siete dimensiones aprovechando tiempos de correlación de rotación cortos.
Se aplicó el método a la proteína de la membrana externa X (OmpX) uniformemente etiquetada con (13) C, (((15) N, desnaturalizada con urea.

Principales resultados:

Se lograron asignaciones de RMN de la columna vertebral y C ((beta) completamente automatizadas y específicas de la secuencia.
Se ha asignado con éxito la proteína OmpX de 148 residuos.
Demostró la eficacia del método para proteínas de hasta 150 residuos.

Conclusiones:

El método automatizado de RMN desarrollado es eficiente y de amplia aplicación.
Esta técnica supera las limitaciones anteriores en el estudio de proteínas desplegadas.
Facilita extensos estudios de resolución atómica de proteínas no globulares desdobladas con desnaturalizantes y funcionales.

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