| The chemistry of biology is complex and there is a great deal that is not understood. A fundamental understanding of biochemical reactions is important to aid in the identification of undiscovered reactions and is critical to fully appreciate the influence that these reactions have on a larger scale. Cellular metal homeostasis exemplifies this requirement. Metal ions are essential cofactors for many enzymes that play vital roles in living organisms, yet accumulation of some metals leads to the catalytic production of toxic species. Therefore, it is essential for cells to regulate metal concentrations. Three general categories of proteins are involved in this process: (1) metal transporters, which are responsible for metal translocation across biological membranes, (2) metallochaperones, which transport metals throughout a cell, and (3) metal regulatory proteins, which respond to metal concentrations to control the cellular levels of the metal transporters and metallochaperones. This work describes the use of Isothermal Titration Calorimetry as a bulk method to quantify the thermodynamics of metal ions binding to metallochaperones (UreE), metal transporters (Irt1 and Fet3p), and other important biological molecules. In addition, novel experimental approaches are described that enable the thorough deconvolution of complex equilibria, data analysis methods are verified using functions that are derived from equilibrium theory to fit data involving a significant amount of speciation, and a methodology is developed to study metal oxidation states that are unstable to oxygen and disproportionation. |
