Characterizing the Binding of Ca2+, Cd2+, and Pb2+ to EF-hand Peptide V of Calbindin D28K and EF-hand Peptides III and IV of Human Cardiac Troponin C Using CD, ITC, and Fluorescence Spectroscopy
Calcium-binding EF-hand proteins are ubiquitous in the cell and are essential for many biological functions such as muscle contraction and cell signaling. Divalent cadmium, Cd2+, and lead, Pb2+ are toxic metal ions that are known to mimic divalent calcium, Ca2+, due to similar ionic radii (100, 95, and 119 pm for Ca2+,Cd2+, and Pb2+ respectively). While Cd2+ and Pb2+ can interact with Ca2+ binding proteins, the affinity and structural changes that occur upon binding are not known. In this study, the following EF-hand peptides were made using solid state peptide synthesis: EF-hand V from Calbindin D28K and EF-hands III and IV from human cardiac troponin C (hcTnC). The peptides were purified by reverse phase HPLC and characterized by MALDI-TOF and QTOF mass spectrometry. The binding of Ca2+, Cd2+, and Pb2+ to these peptides were studied using CD, ITC, and fluorescence spectroscopy. CD structural data revealed that both Cd2+ and Ca2+ bound peptide display enhanced absorption at 220 nm, which is indicative of alpha-helical formation. Pb2+ can also induce structural changes comparable to that of Ca2+ for D28k V and hcTnC IV but elicits some beta sheet formation when bound to hcTnC III. Furthermore, ITC, CD, and fluorescence titrations indicate that these metal ions bind to these peptides with similar affinities. More studies are required to extract condition independent values for direct comparison.
East Carolina University