Toxic Metal Interactions with Human Cardiac Troponin C and Cysteine Replacement Mutants: Investigating Potential Cd(II) Binding to Cysteine Thiols Using DTNB Time Course Assays

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Amanda Fyle (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Calcium binding proteins contribute to all aspects of cell function by binding to calcium directly. Human Cardiac Troponin C (hcTnC) belongs to the EF-hand family of calcium binding proteins. This specific protein is responsible for heart muscle contraction and relaxation. Human cardiac troponin C uses the Ca(II) ion to create a conformational change allowing for correct function to occur. HcTnC is comprised of two domains, the regulatory N-domain and structural C-domain, with each domain containing an EF-hand pair. It has been shown that other heavy divalent metals such as Cd(II) can mimic Ca(II) binding in hcTnC, in turn causing metal toxicity. Previous isothermal titration calorimetry (ITC) and circular dichroism (CD) collected in the Spuches lab revealed that hcTnC protein binds three Cd(II) ions with one in the N-domain and two in the C-domain. In contrast, Ca(II) binds 2 ions with positive cooperativity to the C-domain and one lower affinity ion to the N-domain. Both metal ions induce little structural change in the N-domain of the protein, while significant changes occur in the C-domain upon binding of Ca(II) or Cd(II). While ITC and CD provide useful thermodynamic and secondary structural characterization of Cd(II) binding to hcTnC, these studies do not determine the nature of ligands bound to Cd(II). The divalent Ca(II) and Cd(II) ions have different ligand preferences. Ca(II) prefers oxygen rich aspartic and glutamic acid ligands, while Cd(II) prefers binding to soft thiol containing cysteine residues. There are two cysteine residues located in positions 35 and 84 of the N-domain of the protein. The goal of this project was twofold. We wanted to first verify that our protein was monomeric using both Native Gel Electrophoresis and Size Exclusion Chromatography (SEC). We next wanted to determine if Cd(II) binds to Cys35 and/or Cys84 of hcTnC by using 5,5-dithio-bis-(2-nitrobenzoic acid (DTNB), an organic molecule used to react to and quantitate free thiols in proteins. Our native gels reveal that wild-type and N-terminal hcTnC as well as C35A, C84A, and C35A/C84A all run as monomeric species. SEC of wild-type and N-terminal domain hcTnC also verifies that these species are monomeric albeit with a larger Stokes radius due to the dumbbell shape of Ca(II) binding sensor proteins. Furthermore, stokes radii of apo, Ca(II), and Cd(II) bound proteins were found to be similar suggesting similar structures for all species. Finally, DTNB studies of apo, Ca(II), and Cd(II) bound wild-type and cysteine replacement hcTnC mutant proteins (C35A and C84A) reveal that Cd(II) does not bind to C35 or C84 at stoichiometric ratios, but does interact with C35 (about 20%) at higher molar ratios. This is consistent with earlier ITC and ICP-OES studies of Cd(II) binding to the N-domain of hcTnC indicating that 1.2 Cd(II) ions were bound to the protein. These data enhance our model of Cd(II) binding to hcTnC and suggest that cysteine does not play a large role in binding Cd(II).

Additional Information

Publication

Thesis

Language: English

Date: 2023

Subjects

hcTnC;Cd(II);metal binding

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