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Harnessing environmental Ca2+ for extracellular protein thermostabilization

Citation

Hellinga, Homme; Allert, Malin (2020), Harnessing environmental Ca2+ for extracellular protein thermostabilization, Dryad, Dataset, https://doi.org/10.5061/dryad.hmgqnk9d6

Abstract

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.

Methods

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.

Funding

Corporate Research Agreement - Becton-Dickinson and Duke University, Award: AGR20112333

Corporate Research Agreement - Becton-Dickinson and Duke University, Award: AGR20112333