Abstract
Zea mays L. centrin (Zmcen) is a 20 kDa calcium binding protein also known as caltractin. We used melittin as a simulated target peptide and examined its interaction with Zmcen to understand the structure of Zmcen and the mechanism of interaction with downstream target peptides. The circular dichroism spectrum was used to characterize the typical α-helix structure of Zmcen, and after combining with melittin, the α-helix content of Zmcen changed. Trp residues in melittin were used as fluorescent probes to monitor changes in the conformation of Zmcen upon melittin binding. The Trp residues in melittin gradually shifted from polar environments to nonpolar environments, fluorescence peaks were significantly blueshifted, and the intensity of the fluorescence peak increased. These results showed that Zmcen and melittin combined in a 1 : 1 ratio to form a new complex. The influence of metal ions on binding was also investigated. The combination of Ca2+ and Zmcen helped expose more hydrophobic regions of Zmcen and promoted the binding of Zmcen and melittin. In addition, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was used as a hydrophobic probe to bind to Zmcen and Zmcen occupied the hydrophobic area on the surface of Zmcen, thereby weakening the binding of Zmcen and melittin. The Biacore experiment was used to calculate the equilibrium constant (K D) for the dissociation of Zmcen and melittin. Melittin mainly binds to C-Zmcen but not to N-Zmcen, indicating that the binding site of melittin on Zmcen was mainly at the C-terminus of Zmcen.