Thermodynamic studies of Ca²⁺ binding to human cardiac troponin C
dc.contributor.advisor | Spuches, Anne M. | en_US |
dc.contributor.author | Skowronsky, Rachel Ann | en_US |
dc.contributor.department | Chemistry | en_US |
dc.date.accessioned | 2012-05-20T15:27:21Z | |
dc.date.available | 2013-07-01T11:15:48Z | |
dc.date.issued | 2012 | en_US |
dc.description.abstract | Human Cardiac Troponin C (HcTnC) is an EF hand protein responsible for initiating contraction of the myocardium (heart muscle). The helix-loop-helix motif, characteristic of members of the EF-hand family, allows HcTnC to act as a Ca²⁺ sensor and relays the calcium signal through the thin filament. The binding of Ca²⁺ to the regulatory domain induces a change in HcTnC conformation which modifies subsequent protein-protein interactions. Mutations that alter the calcium sensitivity of HcTnC can lead to cardiomyopathies. One treatment for cardiomyopathies is the use of calcium sensitizing/desensitizing drugs which bind to HcTnC. A thorough understanding of the thermodynamic forces that drive calcium binding to HcTnC and allow it to act as a calcium sensor is crucial for future drug design. Isothermal titration calorimetry (ITC) is a quantitative technique which directly measures the heat of a binding reaction. Calorimetric measurements can be used to obtain the following thermodynamic parameters: binding constant (K), enthalpy (Delta H), stoichiometric ratio (n), enthalpy (Delta S), and Gibbs energy (Delta G) of a wide variety of biochemical reactions including Ca²⁺ binding to proteins. Herein we report the first calorimetric study of Ca²⁺ binding to human cardiac troponin C. Calcium binding isotherms obtained at 25 °°C and at pH 7.0 (10 mM 2-(N-morpholino)ethansulfonic acid (MES), 50 mM KCl) allowed us to obtain thermodynamic parameters for Ca²⁺ ions binding to both the high and low affinity domains. Binding constants are consistent with those reported previously in the literature. Furthermore, binding to the low affinity N-domain was found to be endothermic and entropically driven, a result that is consistent with calcium binding to the regulatory domains of wheat germ calmodulin and the third site of skeletal troponin. Thermograms obtained at two additional temperatures, 10 °C and 37 °C, conducted under identical buffer conditions, allowed the change in heat capacity (Delta C[subscript]p) to be calculated from the slope of Delta H plotted against temperature. The enthalpies of binding for the two events exhibit a trend; as temperature increases the enthalpy becomes more favorable. ITC was also used to determine the thermodynamic parameters associated with Ca²⁺ binding to an isolated N-domain of HcTnC (HcTnC₁₋₈₉). Comparison of the N-domain of HcTnC and the isolated N-domain reveal a favorable free energy of calcium binding to the N-domain when isolated or attached to the C-domain. However, differences in the entropic and enthalpic contributions to the free energy of binding provide supporting evidence for the cooperativity of the N and C-domains. | en_US |
dc.description.degree | M.S. | en_US |
dc.format.extent | 99 p. | en_US |
dc.format.medium | dissertations, academic | en_US |
dc.identifier.uri | http://hdl.handle.net/10342/3909 | |
dc.language.iso | en_US | |
dc.publisher | East Carolina University | en_US |
dc.subject | Chemistry | en_US |
dc.subject | Chemistry, Biochemistry | en_US |
dc.subject | Chemistry, Inorganic | en_US |
dc.subject | Calcium binding | en_US |
dc.subject | Calorimetry | en_US |
dc.subject | EF-hand proteins | en_US |
dc.subject | Human cardiac troponin C | en_US |
dc.subject | ITC | en_US |
dc.subject | Thermodynamics | en_US |
dc.subject | Inorganic chemistry | |
dc.subject | Biochemistry | |
dc.subject.lcsh | Calcium-binding proteins | |
dc.subject.lcsh | Myocardium--Diseases--Treatment | |
dc.subject.lcsh | Calcium | |
dc.subject.lcsh | Proteins | |
dc.title | Thermodynamic studies of Ca²⁺ binding to human cardiac troponin C | en_US |
dc.type | Master's Thesis | en_US |
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