engleski

Influence of engineered disulfide bridge on properties of metal sensing transcription factor MntR from Bacillus subtilis

Transition metals are essential for a variety of biological processes in the living cell: they serve as centres of enzyme catalysis, structural constituents of proteins and stabilizers of protein structure. However, they can be toxic if present in high concentrations. Manganese is an essential metal that is used in bacteria for basic cellular processes such as DNA replication and resistance to oxidative stress. Its cytoplasmic concentration and effect on virulence in pathogens varies greatly between species, but it has been identified as necessary for survival and infection in multiple bacteria. The most common mechanism for regulating metal ion homeostasis in bacteria involves metal sensing transcription factors. These proteins reversibly bind metal ions, which modulate their DNA binding affinity. The main goal of our research is to understand details of the molecular mechanism through which bacterial metallosensors accomplish their role in regulating the concentration of manganese ions. We started our investigations with the manganese- responsive metallosensor MntR from B. subtilis. This protein is a homodimer both in its apo- and metal-bound form, and binds two metal ions per monomer which form a binuclear cluster at the dimer interface. Molecular dynamics simulations have shown that even without metal ion present, DNA binding and dimerization domains of the protein come in close contact (that is usually stabilized by Mn2+). To see if we can stabilize this contact differently, we created a double mutant D8C/E99C of B. subtilis MntR with the goal of establishing disulfide bridge that would replace Mn2+ ion in connecting these domains. Both native and mutant protein were overexpressed in E. coli cells and purified via affinity chromatography and size-exclusion chromatography. Their secondary structure properties and DNA binding properties were determined via CD and DSC measurements. Obtained results are discussed, with aim of better understanding of MntR mechanism of action.

Bacillus subtilis ; metallosensor ; MntR protein ; engineered disulfide bridge ; molecular dynamics simulations ; experimental protein characterization

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