Using biophysical techniques to study the mechanism of ligand-gated potassium efflux systems (KEF) from bacteria

Abstract

The ligand-gated potassium channels KefC and KefB of Escherichia coli are critical components in protecting cells from toxic electrophiles.  Potassium efflux through these channels is coupled to a decrease in cytoplasmic pH which in turn reduces the damage to DNA by electrophiles.  KefC and KefB are both inhibited by cytoplasmic glutathione and activated by glutathione adducts, such as ESG, formed by conjugation of glutathione with electrophilic compounds. Robust membrane purification protocols were developed to isolate both the wild type full-length KefC and KefB and the mutants required for biophysical analysis.  In vivo K+ measurements were performed to ensure that all of the constructs used were fully functional.  Structural and functional analysis used electron paramagnetic resonance (EPR) and stead state emission fluorescence measurements in vivo, on wild type and mutated full-length proteins to elucidate the gating mechanism and test the model generated from crystallographic data.  In particular, EPR spectroscopy combined with site-directed spin labelling revealed a substantial conformational change and thus provided the first insight into coupling between sensing and gating.  Steady state fluorescence spectroscopy was used to precisely measure binding affinities for both activating and inhibitory ligands and characterise nucleotide binding to KefC.  Finally, a variety of chemically diverse glutathione adducts was tested on KefC in vitro to elucidate the mechanism by which these ligands initiate K+ flux through the associated transmembrane domain.EThOS - Electronic Theses Online ServiceGBUnited Kingdo