Pore-forming toxins are fascinating molecules that undergo dramatic conformational changes during their mechanism of action. They are able to form pores, also termed nanopores due to their nanometre size, in lipid membranes. Formation of pores is a stepwise process that involves binding to lipid membranes, oligomerisation at the membrane level and a conformational change that allows part of the polypeptide chain to pass through the lipid membrane. The pores have a diameter of several nanometres and can lead to cell death because of disbalance of crucial ions, loss of nutrients or signalling induced by pores.

We used an actinoporin from the mountainous star coral to create stable pores on the surface of liposomes. Soluble pores were then obtained by extraction from lipid membranes using detergents. Cryo-electron microscopy analysis of the octameric pore structure revealed the presence of membrane lipids forming a complex network of interactions involving both protein-lipid and lipid-lipid interactions. Remarkably, each protomer within the pore interacted with 10 (phospho)lipids and 4 cholesterol molecules. The lipids were found to be essential for the assembly of the pore on the membrane surface and also influenced the functional properties of the pore. Structural analysis and molecular modelling revealed different roles of the lipid molecules in the process of nanopore formation:

i) Receptor lipids initiated the interactions of the protein monomer with the lipid membrane.

ii) Structural lipids were crucial for complex formation and formed an integral part of the final pore complex.

iii) Bridging lipids had minimal contact with the protein but played a role in stabilising monomer-monomer interactions through acyl chain-mediated interactions.

Analysis of the protein-lipid complex highlighted the multiple roles of lipids in both the assembly of macromolecular complexes on the membrane surface and in the stabilisation of the final oligomeric complex.

Nanopores have recently attracted considerable attention due to their successful use in sequencing of DNA, RNA and sensing of proteins. Despite remarkable progress in this field, the availability of unique nanopores is still limited. We focus on developing sensing applications that utilise natural protein-based nanopores and employ high-throughput biophysical approaches. We have convincingly demonstrated that the pores formed by a coral protein can be used to distinguish between variants of highly positively charged proteins.

Este curso persigue como objetivos:

• Dar a conocer las aplicaciones de toxinas con capacidad de formar poros en las membranas celulares.
• Introducción al mecanismo de formación de poros en membranas lipídicas.
• Uso de la criomicroscopía electrónica y modelado molecular para el estudio de interacciones membrana-toxina.
• Uso de nanoporos como biosensores para la diferenciación de proteínas de alta carga positiva.

Conferencia magistral.

Asistencia presencial al curso.