Protein dynamics and DNA binding of native metal-sensing transcription factor MntR from Bacillus subtilis and its D8C/E99C double mutant Ivana Leščić Ašler1, Stephanie Steinberger2, Victor U. Weiss2, Martina Marchetti-Deschmann2, Zoe Jelić Matošević3, Katarina Radman3, Branimir Bertoša3 1 Ruđer Bošković Institute, Division of Physical Chemistry, Bijenička cesta 54, HR-10000 Zagreb, Croatia, ivana.lescic.asler@irb.hr 2 TU Wien, Institute of Chemical Technologies and Analytics, Getreidemarkt 9/164, 1060 Wien, Austria 3 University of Zagreb, Faculty of Science, Department of Chemistry, Horvatovac 102A, HR-10000 Zagreb, Croatia Manganese (II) ions are essential for a variety of bacterial cellular processes, such as DNA replication and resistance to oxidative stress. In the bacterium Bacillus subtilis, Mn2+ ion homeostasis is regulated by the metal-sensing transcription factor MntR. Binding of manganese ions increases the DNA binding affinity of MntR, allowing it to regulate manganese import systems in a concentration dependent way. MntR functions as a homodimer, with each monomer containing a dimerization domain and a DNA-binding domain, and a binuclear metal-binding site (Huang X et al. (2017) Mol Microbiol 103, 253). It has been proposed that one of the bound metal ions is responsible for proper orientation of domains to enable DNA binding. We employed molecular dynamics (MD) simulations to investigate changes in B. subtilis MntR structure and dynamics occurring upon binding of Mn2+. Analyses of MD simulations indicated that the binding of Mn2+ in one of the binding sites keeps the protein domains in closer proximity (Jelić Matošević Z et al. (2023) Int J Mol Sci 24, 957). We attempted to test this by replacing the Mn2+ ion in this metal-binding site with a disulphide bridge between the two protein domains. To this end, we created a double mutant D8C/E99C of B. subtilis MntR. The results of MD simulations showed the behaviour of this mutant to be more similar to the behaviour of Mn2+-bound native protein than the apo-protein. Mutant MntR was purified in the same manner as the native protein (Jelić Matošević Z et al. (2023) Int J Mol Sci 24, 957). The existence of a disulphide bridge was confirmed by Ellman’s reagent reaction. Subsequently, we investigated Mn2+- and DNA-binding of native and mutant MntR by nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA), using a duplex DNA oligomer of known recognition sequence for MntR. Our results have shown that nES GEMMA is a suitable technique for detection of non-covalent protein-DNA complexes, and that native and mutant MntR behave differently in nES GEMMA.