Counting Protons: Using Isothermal Titration Calorimetry to Determine the Number of Protons Exchanged Upon Ca(II) and Cd(II) Binding to Wild-type HcTnC and Mutant C84A and C35A/C84A Proteins

Maintaining a delicate balance of metal ions in the cell is crucial to the survival of many organisms. Divalent cadmium , Cd(II) , is a non-essential metal ion that can disrupt this delicate balance and lead to deleterious effects. Cd(II) imparts its toxicity by mimicking essential metal ions such as Ca(II) and replacing these metals in various proteins. Many methods have been used to characterize both essential and toxic metal interactions with proteins. Isothermal Titration Calorimetry (ITC) is a powerful calorimetric method that measures the heat of a binding reaction. From a single titration experiment , one can obtain all thermodynamic parameters (Ka , [delta]H , [delta]G , and [delta]S) for a binding event. ITC will be used in this study to understand the thermodynamic driving forces that drive Cd(II) binding to Ca(II) binding proteins. The protein of interest is Human Cardiac Troponin C (HcTnC) , an EF-hand protein that requires Ca(II) to regulate heart muscle contraction. In this study , ITC was used to determine the number of protons displaced upon Ca(II) and Cd(II) metal binding to the wild-type , C35A/C84A and C84A mutant proteins. These numbers are required to extract buffer-independent binding parameters which allow for direct comparison between metals and proteins. Most importantly , one can use the number of protons exchanged upon Cd(II) binding to the N-domain of both wild-type and C35A/C84A to shed light on the location of third Cd(II) ion in the N-domain. ITC experiments revealed that Ca(II) displaced a total of 1.1 (± 0.01) and 1.6 (± 0.8) protons in the C- and N-domain of both the C35A/C84A and C84A mutants , respectively. Upon the titration of Cd(II) into the C35A/C84A and C84A mutants , experimental results showed that in the C-domain , a total of 1.1 (± 0.03) and 1.1 (± 0.01) protons were displaced in each mutant respectively. In the N-domain , a total of 0.6 (± 0.1) , 1.6 (± 0.4) and 0.7 (± 0.4) were displaced for the wild-type , C35A/C84A and C84A mutant respectively. It is important to see that the wild-type and C84A were within error of each other. Both the wild-type and C84A contain the cysteine in loop I , which gives indirect evidence that there is a Cd(II) binding to loop I. From the number of protons displaced , the buffer independent values for Ca(II) binding to the C35A/C84A and C84A mutants were determined. The buffer independent parameters (K , [delta]G , [delta]H , and T[delta]S) for Ca(II) binding to the C-domain in both mutants. Buffer independent enthalpy values were obtained for the N-domain of both mutants only. While buffer independent enthalpy values were obtained for Cd(II) binding to both the C- and N-domains of WT , C84A and C35A/C84A , further research is needed to conclude buffer independent binding parameters (K , [delta]G , T[delta]S) for Cd(II) binding to HcTnC and mutants because KITC was far too large to determine accurately