Thermodynamic studies of Ca²⁺ binding to human cardiac troponin C / by Rachel Ann Skowronsky.
| Author/creator | Skowronsky, Rachel Ann |
| Other author | Spuches, Anne M. |
| Other author | East Carolina University. Department of Chemistry. |
| Format | Theses and dissertations |
| Publication Info | [Greenville, N.C.] : East Carolina University, 2012. |
| Description | 99 pages : digital, PDF file |
| Supplemental Content | Access via ScholarShip |
| Subjects |
| Summary | 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. |
| General note | Presented to the faculty of the Department of Chemistry. |
| General note | Advisor: Anne Spuches. |
| General note | Title from PDF t.p. (viewed July 3, 2012). |
| Dissertation note | M.S. East Carolina University 2012. |
| Bibliography note | Includes bibliographical references. |
| Technical details | System requirements: Adobe Reader. |
| Technical details | Mode of access: World Wide Web. |
Availability
| Library | Location | Call Number | Status | Item Actions |
|---|---|---|---|---|
| Electronic Resources | Access Content Online | ✔ Available |