Calcium titration of Chlamydomonas reinhardtii centrin and its structural changes

• 2D IR correlation spectroscopy study has proven useful in the study of CaBP’s.
• Comparative analysis of the molecular behavior of centrin and its terminal domains.
• Centrin behaves as two independent domains.
• The N-terminal domain of centrin governs calcium binding behavior.
• Protein aggregation due to adsorption and the extent of aggregation was estimated.

Chlamydomonas reinhardtii centrin is a highly conserved calcium binding protein belonging to the EF-hand superfamily. Centrin, like other calcium binding proteins, changes conformation upon calcium binding. In addition, the calcium binding sites are comprised mainly of aspartates and glutamates which would serve as probes for a calcium binding event. 2D IR correlation spectroscopy has proven to be a valuable technique to determine the differences in the molecular behavior of the EF-hand domains within centrin. Moreover, the differences in affinity for calcium displayed by these domains were correlated to differences in the molecular behavior of these EF-hand domains when compared with each other and the full-length protein. We were able to confirm the nature of the two independent domains within centrin. Furthermore, we established the mechanism of aggregation was self-association due to adsorption of centrin to the ZnSe ATR crystal and estimated the extent of aggregation of the full-length protein.