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Structural analysis of CLN3: a conserved transmembrane protein with a novel protein fold

Principal Supervisor: Dr Tim Knowles - School of Biosciences

Co-supervisor: Dr. Richard Tuxworth

PhD project title: Structural analysis of CLN3: a conserved transmembrane protein with a novel protein fold

University of Registration: University of Birmingham

Project outline:

Batten disease is a rare, fatal, inherited childhood-onset form of neurodegeneration. Symptoms include vision loss, personality changes and behavioural problems leading to seizures, loss of motor function and eventual death. A mutation in the CLN3 gene has been linked to the development of Batten disease, this gene is highly conserved through evolution but despite more than 25 years of study, its function remains unknown and hence understanding of the biology underpinning the disease is poor. What is clear is the function of the CLN3 protein must also be conserved through evolution because the human CLN3 gene complements disruption of the yeast orthologous gene, Btn1.

The CLN3 protein is a membrane protein predicted to have six transmembrane spans and an amphipathic helix. It has been demonstrated experimentally to exist in cells as a dimer of 12 spans. As many pores, transporters and channels have 12 transmembrane spans, it is likely that Cln3 is also a transporter. However, the primary amino acid sequence shares no homology with any other protein and predictions suggest no proteins sharing a similar structure. This strongly suggests the CLN3 protein has a novel fold. This project aims to identify that fold by solving the structure through expression of the pure protein followed by high resolution cryo electron microscopy and X-ray crystallography.

Expression of multi-subunit transmembrane proteins in fully folded and functional forms is not trivial. We have developed an expression and purification strategy using insect cells and a novel detergent-free nanodisc system invented by Dr. Knowles. The first part of this project will focus on optimising the expression system and testing functionality by ligand binding. Next, a full biophysical characterisation of the purified protein will be performed using techniques including circular dichroism, analytical ultracentrifugation, fluorescence, differential scanning calorimetry, UV spectrophotometry and small angle X-ray scattering to obtain basic information about its structure, aggregation state and stability. This will lead on to state-of-the-art high resolution cryoEM and X-ray crystallography to obtain full structural information.

Disease-associated mutations have been identified throughout the human CLN3 sequence. Once a structure has been solved for wild-type CLN3 we will map the disease-causing variants to the structure to pinpoint key regions for function. If the structure of CLN3 does indeed point to it being a transporter, highlighting the key functional regions will allow predictions of solute size and charge.

In the second part of the project genome editing will be used to rescue Drosophila mutants of CLN3 with wild-type and point mutated forms of the human CLN3 gene. Dr. Tuxworth has identified that Drosophila CLN3 mutants have neurodevelopmental defects in keeping with the early-onset human disease. Drosophila will be used as an in vivo test system to confirm the structural predictions of CLN3 function. Finally we will take wild-type and point mutated forms of CLN3 and express them in Xenopus ooctyes to test likely solute transport and/or ion transport activity based on the structural predictions for cargo.

References:

  1. Ratajczak et al, 2014. FRET-assisted determination of CLN3 membrane topology. PLoS One. Jul 22;9(7):e102593. doi: 10.1371/journal.pone.0102593. eCollection 2014. PMID: 25051496

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

  • Cell culture
  • Protein expression and purification
  • Biophysical techniques
  • CryoEM
  • Genome editing
  • Drosophila genetics
  • Neurobiology
  • Live imaging
  • Electrophysiology
  • Behavioural analysis
  • Xenopus oocyte expression and pharmacology

Contact: Dr Tim Knowles, University of Birmingham