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Caracterización estructural de los complejos de dihidrofolato reductasa mediante espectrometría de masas de
Michael B Cammarata1, Ross Thyer1, Jake Rosenberg1
1†Department of Chemistry and ‡Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, United States.
Journal of the American Chemical Society
|July 1, 2015
Resumen
La espectrometría de masas UVPD de 193 nm rastrea los cambios conformacionales en la dihidrofolato reductasa (DHFR). Esta técnica mapea los sitios de unión de ligando para NADPH y metotrexato (MTX), revelando la flexibilidad de la enzima durante su ciclo catalítico.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Biología Estructural Biología estructural.
- Espectrometría de masas por espectrometría de masas.
Sus antecedentes:
- La dihidrofolato reductasa (DHFR) sufre cambios conformacionales significativos durante la reducción del dihidrofolato a tetrahidrofolato.
- Comprender estos cambios dinámicos es crucial para comprender la función de la enzima y diseñar inhibidores dirigidos.
Objetivo del estudio:
- Para caracterizar el paisaje conformacional de DHFR en presencia del cofactor NADPH y el inhibidor metotrexato (MTX) utilizando 193 nm UVPD.
- Mapear los sitios de unión de NADPH y MTX dentro del DHFR y correlacionar los patrones de fragmentación con los reordenamientos conformacionales.
Principales métodos:
- Se empleó la espectrometría de masas de fotodissociación ultravioleta (UVPD) de 193 nm para analizar complejos binarios (DHFR·NADPH, DHFR·MTX) y ternales (DHFR·NADPH·MTX).
- Se logró una amplia cobertura de la secuencia, produciendo iones fragmentos que indican interacciones de ligando y estados conformacionales.
Principales resultados:
- La UVPD proporcionó una alta cobertura de secuencias (>80%) y reveló fragmentos ligandos, mapeando NADPH al dominio de unión a la adenosina y MTX al dominio del bucle.
- Los patrones de fragmentación mostraron variaciones significativas en las propensiones de escisión de la columna vertebral, lo que refleja cambios conformacionales como la rotación del subdominio y los movimientos de bucle en la unión de ligandos.
- Regiones específicas exhibieron una escisión mejorada (mayor flexibilidad) o una escisión suprimida (protección de ligandos / interacciones intramoleculares).
Conclusiones:
- 193 nm UVPD es una técnica versátil y sensible para el monitoreo de la dinámica de conformación de la enzima durante los ciclos catalíticos.
- El estudio correlacionó con éxito la fragmentación UVPD con los aspectos estructurales y funcionales conocidos de DHFR, validando la utilidad del método.
- Los hallazgos proporcionan información sobre el estado de transición del complejo ternario y la flexibilidad de las enzimas.
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