UCSF

Unlike other bacterial metal ion resistance systems, which either actively transport metal ions out of the cytosol or utilize soluble proteins to sequester metal ions in the periplasm or outside the cell, proteins of prokaryotic mercury resistance (mer) loci confer resistance to inorganic mercury (Hg²⁺) by facilitating its uptake and reduction to elemental mercury (Hg⁰). The expression of both a membrane transporter and mercuric reductase (MerA) defines the minimal set of proteins needed to confer inorganic mercury resistance in prokaryotes. The efficacy of the mer system and of other metal ion resistance pathways is dependent on the specificity, thermodynamics, kinetics and dynamics of the proteins that comprise each system. This dissertation examines some of these properties of the mer proteins and protein domains of MerA and of MerT, the most prevalent membrane transporters in mer isolates. First, we describe work aimed at expressing and purifying MerT for X-ray crystallography studies. By understanding the structure of MerT, we aimed to elucidate the mechanism by which Hg²⁺ is transported into the cell and made available to MerA. Second, we present a novel method for the expression and purification of intact MerA and models fit to small-angle X-ray and neutron scattering observations of MerA in the absence of Hg²⁺ and of an intermediate model of Hg²⁺-handoff from the N-terminal domain (NmerA) and to the catalytic core (Core) of MerA. These are the first structural studies of the linker regions that tethers NmerA to Core. Third, we examine the steady-state kinetics of intact MerA. Here we show that NmerA tethered to Core provides a kinetic advantage in reducing Hg²⁺ when it is associated with either proteinaceous or low molecular-weight ligands. Finally, we introduce evidence that the linker region which tethers NmerA to Core also serves a secondary purpose of localizing MerA to the cell membrane absent of Hg²⁺ and/or MerT.