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Biochemical and biophysical characterization of the manganese transport regulator (MntR) from Bacillus subtilis


Metal ions are employed in biology for several reasons including their ability to participate in redox chemistry, catalysis, and structural stabilization of proteins. However, the properties that make metal ions so widely utilized in biology can be potentially hazardous, particularly if abnormal quantities of these ions are accumulated. This necessitates a mechanism by which the balance between uptake of essential metal ions and efflux of excess essential or toxic metal ions, otherwise referred to as metal homeostasis, can be maintained. Bacteria employ a unique set of metal responsive transcription factors (metalloregulators) to manage this delicate balance. The biochemical and biophysical characterization of MntR, a manganese responsive regulator from the DtxR family is the focus of this thesis. Fluorescence anisotropy was used to probe the DNA-binding of wild type MntR, MntR D8M, and MntR E99C mutants to the cognate DNA recognition sequences mntH and mntA in the presence of various divalent metal ions. Our studies demonstrate the extent to which these metal ions are able to activate MntR to bind DNA. In addition, these studies shed light on the origin of metal specificity between MntR and DtxR and are in agreement with in vivo data reported in the literature. In addition to investigating the DNA- binding abilities of MntR, we also examined the metal binding affinities of this protein in order explain how it fits into the DtxR family and the general field of metalloregulatory proteins. The results demonstrate that MntR metal-binding affinities loosely follow the Irving- Williams series. Interestingly, the protein exhibits the weakest affinity for one of its cognate metal ions. Finally, the metal-mediated mechanism of DNA binding by MntR was studied. Initial investigations using circular dichroism and an environmentally-sensitive dye ANS showed that metal binding stabilizes either tertiary or quaternary structure of MntR. Subsequent studies focused on localizing these structural changes using deuterium exchange mass spectrometry (DXMS) and demonstrated that metal-binding serves to rigidify the pre-organized structure of MntR. Moreover, contrary to typical observation of transcription factors, cofactor (metal) binding does not appear to alter the structure of helix- turn-helix DNA-binding motif

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