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Structural biology of the Ccr4-Not nuclease complex using electron paramagnetic resonance (EPR) spectroscopy and integrative modelling

School of Pharmacy

About the Project

The ultimate goal in molecular biology is to understand normal physiology and disease processes at atomic resolution. Methods such as x-ray crystallography and NMR spectroscopy have been phenomenally successful in the past decades and continue to be the workhorses of structural biology. However, these techniques have limitations, particularly when studying multi-domain proteins and multi-subunit protein complexes or systems with conformational flexibility. The requirement for large amounts of sample can be an additional barrier. While recent progress in cryo-electron microscopy opens up exciting opportunities to study large macromolecular assemblies, there remains a need for additional complementary approaches.

This project aims to apply an integrative structural approach to understand the structure and function of the human Ccr4-Not nuclease complex. This protein complex plays a key role in regulated mRNA degradation and is recruited to mRNAs targeted for degradation and translational repression by the microRNA-machinery, RNA-binding regulatory proteins, or protein recognising RNA methylation (N6-methyladenosine). The protein complex is composed of eight subunits, which are organised into four distinct modules. While the crystal structures of three essential modules are available in the public domain, it is unclear what the relative localisation of these modules is.

To obtain information about the relative location of the modules, we will carry out distance measurements using electron paramagnetic resonance (EPR) spectroscopy. These measurements will be combined with available crystal structures in public repositories and molecular modelling techniques to assemble high-quality structural models. To this end, you will (partially) reconstitute the human Ccr4-Not complex using bacterially expressed proteins based on established procedures in the lab. To facilitate distance measurements, the proteins will be labelled at specific sites using the incorporation of unnatural amino acids that allow the attachment of spin labels using click chemistry. Subsequently, rigid body docking protocols will be used to obtain high-quality structures of the Ccr4-Not nuclease complex.
Preliminary data and established protocols in the lab demonstrate the feasibility of the project.

This is an exciting multidisciplinary project that will provide training in diverse techniques, including an in-depth knowledge of protein structure and RNA degradation pathways, as well as practical skills including DNA cloning, protein expression and purification, site-directed mutagenesis, biochemical assays, biophysical techniques, and computational modelling. You will gain a skillset that provides an excellent foundation for a successful career in academia or industry.

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