The objective of the PhD project was to determine the high-resolution structure of cardiacRyR (RyR2) using single particle cryo-EM. In my PhD work, I first developed a purification strategy for RyR1, reconstituted the ion channel into lipid nanodiscs and performed biochemical and structural characterization of RyR1 in a lipid environment. The structure of reconstituted RyR1 was determined under two conditions that revealed an open channel conformation in the lipid environment. Comparison to published RyR1 structures determined in detergents combined with additional biochemical assays revealed the effect of lipid mimetics on the open probability of the channel. The purified RyR1 was used for the generation of RyR specific nanobodies that were used to develop a novel nanobody-based purification of bovine cardiac RyR isoform, RyR2, in lipid nanodiscs. This fast and reproducible purification with small amounts of starting material has allowed preparation of cryo-EM samples. The intermediate resolution structures of RyR2 were determined with and without the accessory protein FKBP12.6 using cryo-EM. These results pave the way towards structural characterization of RyR2 in complex with small-molecule modulators towards developing efficient drugs targeting the cardiac arrhythmia disorders CPVT1 and ARVD2, which are an important cause of sudden cardiac death in young people.
|Effective start/end date||1/01/14 → 31/12/17|
Flemish discipline codes
- Medical biotechnology diagnostics