Characterizing the Binding of Toxic and Essential Metals to EF-Hand Peptides III and IV of Human Cardiac Troponin C Using Fluorescence Spectroscopy and Isothermal Titration Calorimetry
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Date
2023-12-05
Access
2025-12-01
Authors
Harrison, Mac
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Publisher
East Carolina University
Abstract
Cadmium is one of the most toxic natural and anthropogenic metals found in our environment. Exposure to cadmium is associated with several cardiovascular conditions, neurodegenerative disorders, and cancers. Cadmium’s toxicity is attributed to its ability to mimic and displace essential metals, such as calcium. This is due to their shared charge state, coordination geometry, and similar ionic radii (0.97 Å and 0.99 Å for calcium and cadmium respectively). Calcium-binding proteins are ubiquitous in the cell and are responsible for biological functions such as cell-signaling, muscle contraction, and metal-ion homeostasis. In this study, the thermodynamics of Ca2+ and Cd2+ binding to EF-hand peptides from the C-domain of human cardiac troponin C (hcTnC), a calcium binding protein responsible for heart muscle contraction, have been determined. Specifically, spectrofluorometric titrations were performed on individual peptides EF-hand III and EF-hand IV to determine metal binding constants. Similarly, isothermal titration calorimetry (ITC) experiments and spectrofluorometric titrations were performed on EF-hand III and EF-hand IV mixed-species peptides to determine metal binding constants as well as thermodynamic parameters such as ΔG, ΔH, and TΔS. Binding parameters were obtained by fitting data to a modified two-step model that assumes the formation of 1:2 metal-peptide complex followed by the addition of a second metal to form the 2:2 dimer species. The best fit association constants, Ka, of data obtained from both instrumental techniques were consistent with one another thus providing a high level of confidence in our model. The Ka for the formation of the 1:2 metal-peptide complex was 106 M for all dimer species, while binding of the second metal ion to form the 2:2 species was approximately 104 M for all dimer species. ITC studies of heterodimer peptides show that entropy is the driving force for metal-binding for both the 1:2 and 2:2 species and that binding occurs with negative cooperativity. Binding of Cd2+ to the C-domain of hcTnC also occurs with negative cooperativity while it is commonly known that Ca2+ binds to EF-hand proteins with positive cooperativity. The results of these studies provide a fundamental thermodynamic framework for understanding essential and heavy metal interactions with EF-hand peptides.