Crystallization of Centrin Complexes

Centrin, a highly conserved calcium binding protein in higher eukaryotes, belongs to the EF-hand superfamily and contains two independent structural domains. It is a ubiquitous component of centrioles and mitotic spindle poles, associated with the microtubule organizing center in all eukaryotes. When centrin is associated with Sfi1 (a fibrous protein that centrin interacts with), is phosphorylated and signals the separation of the centrosome. By using a centrin variant, hCen1 (E105K), we want to prove that if a salt bridge that is thought to stabilize the hCen-Sfi1 is disrupted, the complex will be destabilized and in turn cause an arrest in the cell cycle. Crystallization of the complexes is needed to know their structure and relate it to its function (or possible effect) in the organism. First, purification of centrin was carried out by incubation at 50°C for 30 min., sonication, centrifugation, and affinity and anion exchange chromatographies. GST-Sfi1 was purified using GST affinity and anion exchange chromatographies. Then, these purified samples were used for crystallization to study the complexes structures but, because optimal crystallization conditions for them were for the most part unknown, crystal screenings were carried out to obtain information on the optimal conditions for these complexes to crystallize.