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Reconstruyendo espectros de RMN de estados de proteínas excitadas "invisibles" utilizando experimentos HSQC y HMQC

Nikolai R Skrynnikov¹, Frederick W Dahlquist, Lewis E Kay

¹Protein Engineering Network Center of Excellence, Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8. nikolai@purdue.edu

Journal of the American Chemical Society

|October 10, 2002

Resumen

Las mediciones de relajación Carr-Purcell-Meiboom-Gill (CPMG) revelan dinámicas de intercambio químico a una escala de tiempo de milisegundos. Un nuevo método determina el signo de las diferencias de desplazamiento químico, crucial para comprender los cambios conformacionales de las proteínas.

Área de la Ciencia:

Química biofísica y bioquímica.
Biología Estructural Biología estructural.
Espectroscopia de Resonancia Magnética Nuclear (RMN) Espectroscopia de Resonancia Magnética Nuclear (RMN) Espectroscopia de Resonancia Magnética Nuclear (RMN) Espectroscopia de Resonancia Magnética Nuclear (RMN) Espectroscopia de Resonancia Magnética Nuclear (RMN) Espectroscopia de Resonancia Magnética Nuclear (RMN) Espectroscopia de Resonancia Magnética Nuclear (RMN) Espectroscopia de Resonancia Magnética Nuclear (RMN) Espectroscopia de Resonancia Magnética Nuclear (RMN) Espectroscopia de Resonancia Magnética Nuclear (RMN) Espectroscopia de Resonancia Magnética Nuclear (RMN) Espectroscopia de Resonancia Magnética Nuclear (RMN)

Sus antecedentes:

La dispersión de relajación de Carr-Purcell-Meiboom-Gill (CPMG) es vital para estudiar el intercambio químico a una escala de tiempo de milisegundos.
Los experimentos de CPMG arrojan tasas de interconversión, poblaciones y diferencias de desplazamiento químico.
El signo de Δω no se puede obtener directamente de los experimentos estándar de CPMG.

Objetivo del estudio:

Desarrollar un método para determinar el signo de las diferencias de desplazamiento químico en los sistemas de intercambio.
Para mejorar la información obtenida de los estudios de dispersión de relajación de CPMG.
Para aplicar la metodología a un sistema de proteínas que exhiben intercambio conformacional.

Principales métodos:

Utilizó mediciones de relajación Carr-Purcell-Meiboom-Gill (CPMG) con espaciado de pulso variable.
Comparó los cambios químicos en las dimensiones indirectas de los espectros HSQC en diferentes campos magnéticos.
Espectro combinado de HSQC y HMQC en un solo campo para determinar el signo de Δω.
Aplicó el método a un mutante de la cavidad de la lisozima T4 (L99A).

Principales resultados:

Determinó con éxito el signo de diferencias de desplazamiento químico (Δω) además de su valor absoluto.
Intercambio conformacional caracterizado en una escala de tiempo de milisegundos en un mutante T4 de la lisozima L99A.
Cuantificó el tipo de cambio en aproximadamente 1450 s-1 a 25°C.

Conclusiones:

El método desarrollado proporciona de manera confiable la señal de las diferencias de desplazamiento químico, complementando los datos de CPMG.
Este enfoque avanza significativamente en la caracterización de los procesos dinámicos en las macromoléculas biológicas.
La determinación precisa de los parámetros de intercambio ayuda a comprender la función y la dinámica de las proteínas.

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