Figure 3.

Grafting approach to probe site-specific metal binding properties of Ca²⁺-binding proteins. (a) Schematic representation of the grafting approach. Any predicted linear Ca²⁺-binding sequence can be inserted into the host protein CD2 domain 1 (CD2.D1) between residues S52 and G53 without disrupting the integrity of the host protein. Metal binding to the engineered protein can be monitored by taking advantage of a potential LRET pair, the buried Trp (W32) within the two layers of beta-sheets and the terbium ion bound to the inserted sequence. (b) Flow chart showing the application of grafting approach to confirm metal binding of predicted Ca²⁺ binding sites. Adapted with permission from ref. [23, 29].