Allosteric Coupling Between Transition Metal-Binding Sites in Homooligomeric Metal Sensor Proteins

Abstract

Intracellular concentrations of transition metal ions are controlled at the transcriptional level by a panel of metalloregulatory proteins that collectively allow the cell to respond to changes in bioavailable metal concentration to elicit the appropriate cellular response, e.g., upregulation of genes coding for metal export or detoxification proteins in the event of metal excess. These proteins represent a specialized class of allosteric regulators that are ideal for studying ligand-mediated allostery in a comprehensive way due to the size, stability, reactivity, and the spectroscopic properties of transition metal ions as allosteric ligands. In addition to the commonly studied heterotropic regulation of metal binding and DNA binding, many of these proteins exhibit homotropic allostery, i.e., communication between two or more identical metal (ligand) binding sites on an oligomer. This chapter aims to guide the reader through the design and execution of experiments that allow quantification of the thermodynamic driving forces (ΔG (C), ΔH (C), and ΔS (C)) that govern both homotropic and heterotropic allosteric interactions in metal sensor proteins as well as the steps required to remove the influence of complex speciation from the measured parameter values.