Ca²⁺ is the third-most prevalent metal ion in the environment.  EF hands are common Ca²⁺-binding motifs found in both extracellular and intracellular proteins of eukaryotes and prokaryotes.   Cytoplasmic EF hand proteins often mediate allosteric control of signal transduction pathway components in response to intracellular Ca²⁺ concentration fluctuations by coupling Ca²⁺ binding to changes in protein structure.  We show that an extracellular structural Ca²⁺-binding site mediates protein thermostabilization by such conformational coupling as well.  Binding Ca²⁺ to the EF hand of the extracellular (periplasmic) Escherichia coli glucose-galactose binding protein thermostabilizes this protein by ~17K relative to its Ca²⁺-free form.  Using statistical thermodynamic analysis of a fluorescent conjugate of ecGGBP that reports simultaneously on ligand binding and multiple conformational states, we found that its Ca²⁺-mediated stabilization is determined by conformational coupling mechanisms in two independent conformational exchange reactions.  Binding to folded and unfolded states determines the maximum Ca²⁺-mediated stability.  A disorder®order transition accompanies formation of the Ca²⁺ complex in the folded state and dictates the minimum Ca²⁺ concentration at which the Ca²⁺-bound state becomes dominant.  Similar transitions also encode the structural changes necessary for Ca²⁺-mediated control elements in signal transduction pathways.  Ca²⁺-mediated thermostabilization and allosteric control therefore share a fundamental conformational coupling mechanism, which may have implications for the evolution of EF hands.

Multi-dimensional fluorescence data of Acrylodan conjugates of the Escherichia coli glucose-galactose binding protein collected on a Roche LightCycler as a function of temperature, glucose and calcium.  Data was analyzed using the Nested Equilibrated States Tree statistical thermodynamics method.  This repository contains the original data and Python scripts for their analysis.