Publication: Nanodiscos como herramienta para estudiar interacciones entre proteínas de división bacteriana
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2014-12-15
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Universidad Complutense de Madrid
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Abstract
La división celular en E. coli implica el ensamblaje de un complejo macromolecular, denominado divisoma, formado por varias proteínas, 10 de ellas esenciales. Se conoce la secuencia lineal y concertada de ensamblaje que siguen estas proteínas del divisoma y algunas de sus actividades bioquímicas, así como algunas interacciones proteína-proteína involucradas. Inicialmente, 3 proteínas, FtsZ, ZipA y FtsA ensamblan juntas formando el proto-anillo en el cual se van incorporando el resto de componentes. En la presente tesis se ha usado un sistema de membrana in vitro, llamado nanodiscos, para estudiar 2 proteínas de división de membrana, ZipA y FtsN. ZipA es una proteína de 36.4 kDa con una única hélice transmembrana seguida de un largo dominio desestructurado flexible y un pequeño dominio C-terminal que une FtsZ en el citoplasma. FtsN, en cambio, presenta una topología inversa, con un pequeño N-terminal desestructurado y cargado en el citoplasma seguido de un dominio transmembrana, un dominio grande y flexible y un pequeño dominio SPOR en la cara periplásmica que interacciona con peptidoglicano. Ambas proteínas se incorporaron en nanodiscos, un modelo de membrana que consiste en un parche de lípidos con forma de disco formando una bicapa rodeado por 2 copias de la proteína MSP (Membrane Scaffold Protein), análoga de la proteína apoA-1, la cual estabiliza la membrana. Estas estructuras son solubles, monodispersas y tienen un diámetro de unos 10 nm. Los nanodiscos proporcionan un entorno topológicamente restringido a las proteínas de membrana mientras las mantiene en solución, permitiendo el uso de un gran número de de herramientas bioquímicas y biofísicas para caracterizar cuantitativamente las proteínas insertadas en ellos y sus interacciones. El objetivo general de esta tesis fue poner a punto el sistema de nanodiscos como soporte para el estudio de interacciones entre proteínas del divisoma de E. coli. Este trabajo es pionero en España, tanto en el uso del sistema de nanodiscos para insertar proteínas de membrana, como en la aplicación de este sistema para medir interacciones entre proteínas de forma cuantitativa
In Escherichia coli, cell division involves the assembly of a macromolecular complex, the divisome, formed by several proteins, 10 of them essential. The nature of a large part of the divisome components is known, as it is their assembly pathway and some of the biochemical activities and protein-protein interactions involved. The elements of the divisome follow an assembly pathway in which both sequential and concerted stages are involved. Initially, three proteins, FtsZ, FtsA, and ZipA, assemble together, forming a proto-ring into which the other components are added. Initial complex subsequently recruits the rest of division proteins, all of them membrane proteins. In this thesis we have used an in vitro membrane model, nanodiscs, to study two membrane-attached division proteins, ZipA and FtsN. ZipA is a 36.4 kDa protein with a single, N-terminal transmembrane helix followed by a large, flexible domain and small C-terminal globular domain that binds to FtsZ in the cytoplasm. On the other hand, has a reversed topology, with small N-terminal unstructured and charged domain in the cytoplasm, followed by a single transmembrane domain, a large flexible domain and a small SPOR domain in the periplasmic that interacts with peptidoglycan. As stated above, both proteins were incorporated into nanodiscs, a membrane model consisting of a discoidal patch of lipid bilayer surrounded by two copies of a Membrane Scaffold Protein (MSP), an apoA-I analogue, which stabilize the membrane. The whole structure is water-soluble, monodisperse, and has a diameter of 10 nm. Nanodiscs provide a topologically restricted environment to membrane proteins while remaining in solution, allowing the use of a broad arsenal of biochemical and biophysical tools to quantitatively characterize the embedded protein and its interactions. The main objective of this thesis was to get ready the nanodisc system as support for the study of interactions between proteins of E. coli divisome. This is a pioneering work in Spain, both in the use of nanodisc system to insert membrane proteins and in the application of this system to measure protein interactions quantitatively.
In Escherichia coli, cell division involves the assembly of a macromolecular complex, the divisome, formed by several proteins, 10 of them essential. The nature of a large part of the divisome components is known, as it is their assembly pathway and some of the biochemical activities and protein-protein interactions involved. The elements of the divisome follow an assembly pathway in which both sequential and concerted stages are involved. Initially, three proteins, FtsZ, FtsA, and ZipA, assemble together, forming a proto-ring into which the other components are added. Initial complex subsequently recruits the rest of division proteins, all of them membrane proteins. In this thesis we have used an in vitro membrane model, nanodiscs, to study two membrane-attached division proteins, ZipA and FtsN. ZipA is a 36.4 kDa protein with a single, N-terminal transmembrane helix followed by a large, flexible domain and small C-terminal globular domain that binds to FtsZ in the cytoplasm. On the other hand, has a reversed topology, with small N-terminal unstructured and charged domain in the cytoplasm, followed by a single transmembrane domain, a large flexible domain and a small SPOR domain in the periplasmic that interacts with peptidoglycan. As stated above, both proteins were incorporated into nanodiscs, a membrane model consisting of a discoidal patch of lipid bilayer surrounded by two copies of a Membrane Scaffold Protein (MSP), an apoA-I analogue, which stabilize the membrane. The whole structure is water-soluble, monodisperse, and has a diameter of 10 nm. Nanodiscs provide a topologically restricted environment to membrane proteins while remaining in solution, allowing the use of a broad arsenal of biochemical and biophysical tools to quantitatively characterize the embedded protein and its interactions. The main objective of this thesis was to get ready the nanodisc system as support for the study of interactions between proteins of E. coli divisome. This is a pioneering work in Spain, both in the use of nanodisc system to insert membrane proteins and in the application of this system to measure protein interactions quantitatively.
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular I, leída el 28-04-2014