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El oxígeno como una sonda paramagnética de agrupación y exposición al disolvente en estados plegados y desplegados de
Irina Bezsonova1, Ferenc Evanics, Joseph A Marsh
1Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON, Canada M5S 1A8.
Journal of the American Chemical Society
|January 27, 2007
Resumen
El oxígeno disuelto revela la dinámica de despliegue de las proteínas. Utilizando desplazamientos paramagnéticos, los investigadores mapearon los cambios en la exposición al disolvente en la proteína Drk.
Área de la Ciencia:
- Bioquímica y Biología Molecular.
- 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:
- El dominio SH3 N-terminal de la proteína Drk de Drosophila (homólogo de Drosophila Nck) existe en un equilibrio de intercambio lento entre los estados plegado (Fexch) y desplegado (Uexch).
- Comprender la dinámica de las proteínas y la accesibilidad del disolvente en estos estados es crucial para descifrar la función de las proteínas y el mal plegamiento.
- Los métodos convencionales de RMN pueden ser limitados en la caracterización de estructuras transitorias o sistemas desordenados.
Objetivo del estudio:
- Para investigar la exposición al disolvente de los estados Fexch y Uexch de la proteína Drk.
- Para utilizar el oxígeno disuelto como una sonda paramagnética para el mapeo de la accesibilidad de disolventes de resolución atómica.
- Evaluar la utilidad de los desplazamientos paramagnéticos de la RMN 13C en el estudio de las estructuras transitorias de las proteínas.
Principales métodos:
- Empleando la espectroscopia de RMN 1H,13C HSQC (coherencia cuántica única heteronuclear).
- Introducir oxígeno disuelto a presiones parciales controladas (30 atm y 60 atm) para inducir cambios paramagnéticos.
- Analizando los desplazamientos paramagnéticos del 13C, normalizados con respecto a los aminoácidos libres, para cuantificar el contacto con el disolvente en los estados plegado y desplegado.
Principales resultados:
- Las diferencias significativas en los desplazamientos paramagnéticos entre los estados de Fexch y Uexch indican perfiles de exposición a disolventes distintos.
- En el estado de Fexch, los residuos del núcleo hidrofóbico mostraron desplazamientos mínimos, mientras que los residuos expuestos a la superficie exhibieron desplazamientos más grandes.
- El estado de Uexch mostró menos variación en los desplazamientos, pero identificó regiones protegidas (por ejemplo, Phe9, Thr12, Ala13, Lys21, Thr22, Ile24, Ile27, Arg38) con estructura residual.
Conclusiones:
- Los desplazamientos paramagnéticos inducidos por oxígeno disuelto proporcionan una poderosa herramienta para mapear la accesibilidad del disolvente con resolución atómica.
- Este método distingue eficazmente los cambios en la exposición al disolvente durante el despliegue de las proteínas.
- Los desplazamientos paramagnéticos son muy valiosos para el estudio de estructuras transitorias y sistemas desordenados donde las técnicas tradicionales de RMN son menos informativas.
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