Un mapa de puntos calientes del complejo ternario FKBP12-Rapamicina-FRB mediante el etiquetado de fotoafinidad y la proteómica basada en la espectrometría de masas
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Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry
Published on: June 8, 2022
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Hope A Flaxman1, Chia-Fu Chang1, Hung-Yi Wu1
1Department of Chemistry and Chemical Biology , Harvard University , 12 Oxford Street , Cambridge , Massachusetts 02138 , United States.
Journal of the American Chemical Society
|July 17, 2019
Resumen
Este estudio extiende el etiquetado de fotoafinidad (PAL) y la espectrometría de masas (MS) para mapear los sitios de unión de moléculas complejas como la rapamicina. Este método revela nuevos modos de unión y objetivos potenciales en el proteoma.
Área de la Ciencia:
- Biología Química
- Proteomía
- Biología estructural
Sus antecedentes:
- El etiquetado por fotoafinidad (PAL) junto con la espectrometría de masas (MS) permite la caracterización estructural de los sitios de unión de moléculas pequeñas.
- Los métodos actuales se limitan a moléculas pequeñas simples, lo que dificulta el estudio de compuestos complejos.
- Comprender las interacciones complejas de las moléculas es crucial para el descubrimiento de fármacos y la identificación de objetivos.
Objetivo del estudio:
- Ampliar los métodos PAL y MS para mapear los sitios de unión de moléculas complejas, específicamente el inmunosupresor rapamicina.
- Definir estructuralmente nuevos modos de unión de los productos naturales macrocíclicos dentro del proteoma.
- Para extraer mediciones estructurales precisas para la interpretación de los datos PAL.
Principales métodos:
- Desarrollo de una sonda de foto-rapamicina con un grupo de diazirina para PAL.
- Formación y análisis del complejo ternario FKBP12-foto-rapamicina-FRB.
- Análisis basado en la espectrometría de masas de complejos digeridos irradiados fotográficamente para identificar los sitios de conjugación.
Principales resultados:
- Aplicación exitosa de PAL y MS para mapear los sitios de unión de la rapamicina, un producto natural macrocíclico complejo.
- Identificación de sitios específicos de conjugación en las proteínas FKBP12 y FRB mediante foto-rapamicina.
- El modelado molecular proporcionó restricciones de distancia y radio de etiquetado para la sonda de foto-rapamicina.
Conclusiones:
- El mapeo de puntos de acceso vinculantes puede aplicarse con éxito a productos naturales macrocíclicos complejos.
- El método desarrollado proporciona mediciones estructurales precisas para interpretar los datos PAL.
- Este enfoque puede permitir el descubrimiento de nuevos sitios de unión para objetivos proteomáticos "indruggables".
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