| Calreticulin is a Ca2+-binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In the ER lumen, Calreticulin cooperates with another chaperone, its close homolog, Calnexin and a thiol oxidoreductase, ERp57 in the "quality control system" during the protein folding process, ERp57 binds to the tip of Calreticulin P-domain. In my thesis, I have used site-specific mutagenesis to change amino acid residues in the P-domain repeat regions that may be involved in interactions with ERp57. Mutations, substitutions into alanines, were designed to disrupt the characteristic beta-sheet structures within the P-domain. Purified wild type Calreticulin and P-domain mutant proteins were examined using standard bio-molecular assays including limited trypsin digestion and intrinsic fluorescence to determine significant conformational changes in mutated proteins comparing to wild type Calreticulin. Malate dehydrogenase (MOH) thermal aggregation assay results suggest that P-domain repeat regions mutations do not have any significant impact on the chaperone function of Calreticulin. Surface plasmon resonance analysis revealed that two particular mutations in P-domain repeat regions: 207KID209-AAA and 189KKIK192-AAAA, significantly enhanced the ERp57 binding capacity of Calreticulin suggesting that those regions are involved in interactions between those two proteins. |
