The first week of research is now done! This week has laid the groundwork for this project. i largely been spent understanding the theory behind the project, the context and experimental procedures to be undertaken.
1- What is the project?
This project revolves around the Connexin 32 protein, seen below;
Sourced from: https://alphafold.ebi.ac.uk/entry/P08033
This protein is a member of the Beta connexin family, of which the members are CO2-sensitive. In the case of Connexin 32 (which I will refer to as Cx32 for brevity) this is mainly by virtue of residues Lysine 104 and 124. As can be seen in the diagram below, when CO2 binds to the K124 residue via a labile bond, which is directed toward the K104 residue by the flanking residues. This CO2 binding creates a 'carbamate bridge' between adjacent subunits that biases the hemichannel to its open state. Once in its open state, molecules such as ATP are then permitted to leave the cell. Unlike other Beta-Connexins, Cx32 is repoted to be potentiated by PCO2 around 70mmHg. These levels are not and cannot be sustained systemically in the body. This project seeks to resolve this discrepancy by testing an addition to the CO2 gating model of Cx32, whereby suggest that Cx32 localises to paranodes of Schwann cells and the nodes of nerves to take advantage of the enhanced CO2 production.
Mutation in the GJB1 gene, which encodes Cx32, result in form of muscular dystrophy called CMTX. The mutations i intend to introduce into Cx32 also provide the chance to assess how mutations in Cx32 lead to CMTX. This will be the primary line of questioning I will explore in this research. My primary aim, via introducing these mutations into specific residues involved in CO2 sensing, is to confirm that Cx32 is gated by 70mmHg and to assess how gating is lost in mutations that mimick CMTX.
What experimental methods will be used?
The primary experimental method to be used that I have not previously encountered is Gibson assembly. As the below diagram shows, to introduce the specific mutations I intend I will make multiple fragments and use the Gibson assembly method to stitch them together in the correct order.
Why is this Project important?
I also spent this week researching the context behind this project. To me it is important that the research i have provides positive societal impact. As stated, mutations in the GJB1 gene, encoding Cx32, induce a form of muscular dystrophy labelled CMTX. I intend to learn more about how the intorduction of these mutations alter the behaviour of the Cx32 protein, which leads to onset of the pathology. There is currently no cure for CMTX, so i believe research such as this is necessary to lay the groundwork and begin the progress toward better treatment or a potential cure.
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