Role of calcium binding in protein structural changes and phospholipid-protein interactions studied by capillary isoelectric focusing with whole column imaging detection

A variety of biochemical and physical properties of proteins are regulated by calcium ion (Ca2+) binding with varying specificity and affinity. Calcium ion binding can adjust the phospholipid-protein interactions through changing the properties of phospholipid membrane. As an attractive detection technique, whole column imaging detection (WCID) coupled to capillary isoelectric focusing (cIEF) displays several advantages in the study of protein-ligand and protein-protein interactions, including fast and high-efficient separation, high resolution, and simple operation. In this study, a cIEF-WCID method was evaluated for studying the effect of Ca2+ binding on protein structural changes and phospholipid-protein interactions. Four proteins with different isoelectirc point (pI), trypsin inhibitor (pI = 4.5), β-lactoglobulin B (pI = 5.2), phosphorylase b (pI = 6.3), and trypsinogen (pI = 9.3), were used for this purpose. The targeted proteins exhibited altered cIEF profiles due to protein conformation changes resulting from the Ca2+ binding. The study showed that Ca2+ can be buried in the phospholipid membrane, modify the membrane property, and change the phospholipid-protein interactions. The utility of the cIEF-WCID technique demonstrates that the calcium binding plays a crucial role in the protein structural changes and the phospholipid-protein interactions, and elucidates the possible mechanisms involved in calcium-protein binding and calcium bound phospholipid-protein interactions.