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Los conjuntos conformacionales basados en la RMN explican la apertura y el cierre de pH del canal OmpG

Tiandi Zhuang¹, Christina Chisholm, Min Chen

¹Department of Molecular Physiology and Biological Physics and Center for Membrane Biology, University of Virginia , Charlottesville, Virginia 22903, United States.

Journal of the American Chemical Society

|September 12, 2013

Resumen

La proteína G de la membrana externa (OmpG) utiliza un mecanismo de pH para el transporte. Nuevos métodos de RMN revelan movimientos de bucle dinámico que controlan el canal OmpG, ofreciendo información sobre la regulación de las proteínas de la membrana.

Área de la Ciencia:

Biología estructural Biología estructural.
La biofísica es la biofísica.
Biología molecular La biología molecular.

Sus antecedentes:

La proteína de la membrana externa G (OmpG) de Escherichia coli es una proteína monomérica beta-barril.
OmpG funciona como una porina inespecífica, facilitando la absorción de los oligosacáridos.
Estudios anteriores sugirieron un mecanismo de poros con pH basado en estructuras cristalinas, con un bucle 6 que bloquea el poro a pH bajo.

Objetivo del estudio:

Para visualizar la dinámica conformacional del bucle 6 de OmpG utilizando RMN.
Para calcular conjuntos conformacionales que explican el mecanismo de los poros por pH.
Para validar los hallazgos utilizando enlaces cruzados de disulfuro y electrofisiología.

Principales métodos:

Espectroscopia de resonancia magnética nuclear (RMN) mejoramiento de la relajación paramagnética (PRE). espectroscopia de resonancia magnética nuclear (RMN).
Cálculo computacional de conjuntos conformacionales.
Experimentos de enlaces cruzados de disulfuro.
Grabaciones electrofisiológicas de un solo canal.

Principales resultados:

El PRE de RMN visualizó con éxito la dinámica conformacional del bucle 6 de OmpG.
Se calcularon conjuntos de conformación, explicando la apertura y cierre pH-gated del canal OmpG.
El enlace cruzado de disulfuro y la electrofisiología validaron los conformadores de bucle derivados de PRE.
Se identificaron conjuntos conformacionales adicionales asociados a la membrana a pH 6.3 y 7.0.

Conclusiones:

El portal del canal OmpG está regulado de manera más dinámica de lo que se suponía anteriormente.
El enfoque NMR PRE es efectivo para caracterizar conjuntos conformacionales funcionalmente importantes en proteínas de membrana.
Esta metodología puede aplicarse ampliamente a otras proteínas de membrana.

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