The ryanodine receptor (RyR) is a large (563 kDa) tetrameric calcium channel that controls the efflux of calcium ions from the large internal stores within myofibrils called the sarcoplasmic reticulum. The cytoplasmic domain is connected to the pore via an "interacting-domain" (I domain) which plays an important role in the mechanism of channel regulation. Within the I domain is a calcium binding region which resembles a lobe of calmodulin and is thus called the calmodulin-like-domain (CaMLD). CaMLD has a binding partner region, the calmodulin-binding-domain (CaMBD) located in the cytoplasmic domain. Mutations in the cardiac isoform of RyR have been shown to result in the clinical presentation of a lethal disease condition called catecholaminergic polymorphic ventricular tachycardia (CPVT). Thus, mutations proximal to the CaMLD and CaMBD region are of particular interest, since if these mutations affect the binding of calcium or result in any conformational change of the protein, then the role of the mutation could be explained. Computer simulation methods were employed to investigate the role of CaMLD and CaMBD in the overall biology of RyR. The individual domains and also the entire section of RyR containing both domains were modelled. This latter model underwent a simulated folding process to generate a tool to further investigate the role of CPVT mutations within this region in the context of the structural changes brought about by the changes in amino acid residues of the protein. CaMLD and CaMBD were produced as recombinant proteins and selected CaMLD CPVT mutants generated. The calcium dependent interaction of these two domains was examined through a range of functional techniques, in particular circular dichroism. All the studied mutants displayed a calcium induced conformational change associated with the functional protein. However, one particular mutation was identified which appeared to significantly weaken the structural stability and have reduced calcium sensitivity when compared to the wild type recombinant protein.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:611020 |
| Date | January 2014 |
| Creators | Wilson, Peter William |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/56964/ |
Page generated in 0.0022 seconds