Characterizing the Binding of Ca2+ and Cd2+ 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
Loading...
Date
8/5/2020
Access
Authors
Taylor, Whitney Cameron
Journal Title
Journal ISSN
Volume Title
Publisher
East Carolina University
Abstract
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.