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Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example
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Las perturbaciones de los aminoácidos aromáticos están asociadas con el ensamblaje de racimos de hierro en la
Adam R Offenbacher1, Jun Chen, Bridgette A Barry
1Department of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
Journal of the American Chemical Society
|April 14, 2011
Resumen
Este estudio utiliza la espectroscopia Raman de resonancia UV para revelar cambios estructurales en las ribonucleótidos reductasas (RNR). Detalla cómo el ensamblaje de racimos de hierro y la reducción del radical tirosilo afectan a los aminoácidos aromáticos, proporcionando información sobre la regulación de las enzimas.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- La espectroscopia es una técnica de espectroscopia.
- Enzimología Enzimología.
Sus antecedentes:
- Las ribonucleótidos reductasas de clase Ia (RNR) son enzimas esenciales que contienen un racimo de diirón y un radical tirosílico.
- Comprender la dinámica estructural de la RNR es crucial para elucidar la función y la regulación de las enzimas.
- Los aminoácidos aromáticos juegan un papel clave en la estructura y actividad de las enzimas.
Objetivo del estudio:
- Para investigar los cambios estructurales en la subunidad RNR β2 inducidos por el ensamblaje de clúster de hierro.
- Para caracterizar las alteraciones estructurales asociadas con la reducción del radical tirosilo (Y122•).
- Explorar las funciones de los aminoácidos aromáticos (tirosina, triptófano, histidina) en la estructura y función de la RNR.
Principales métodos:
- Se empleó la espectroscopia de diferencias de resonancia ultravioleta Raman (UVRR).
- Los espectros de UVRR se mejoraron a 229 nm, probando las contribuciones de aminoácidos aromáticos.
- El etiquetado isotópico y las comparaciones de compuestos modelo se utilizaron para las asignaciones espectrales.
Principales resultados:
- La reducción de radicales de tirosilo produjo bandas de Raman específicas para Y122• y Y122, con una mínima perturbación de otros residuos aromáticos.
- El ensamblaje de clúster de hierro indujo la desprotonación de histidina, respaldada por estudios de titulación p(2) H que muestran un pK elevado.
- Se observaron perturbaciones en la tirosina y el triptófano; los cambios en el triptófano sugieren un aumento de la unión de hidrógeno y una conformación alterada cerca del centro del metal.
Conclusiones:
- Los cambios electrostáticos y conformacionales en los aminoácidos aromáticos acompañan al ensamblaje de grupos metálicos en RNR.
- Los residuos de triptófano perturbados se encuentran en un entorno protegido de baja dieléctrica cerca del centro del metal.
- Es probable que estos cambios conformacionales contribuyan a la regulación alostérica de la unión de metales en RNR.
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