Effect of chemical denaturants on the conformational stability of GyrB subunit of DNA gyrase from Salmonella enterica serovar Typhi.

DNA gyrase, a type II topoisomerase maintains the topology of DNA by introducing negative supercoils using energy generated by ATP hydrolysis. It is composed of two subunits, GyrA and GyrB (GyrA2 GyrB2 hetero-tetramer). GyrB comprises two domains, a 43kDa amino N-terminus (GBNTD) and 47kDa carboxyl C- terminus (GBCTD). Till now no study has been reported in terms of stability of Gyrase B and its domains using chemical denaturants related to its function. To understand the role of each domain in GyrB subunit, we estimated the thermodynamic stability of GBF and its individual domains using urea and GdmCl. Changes in secondary and tertiary structures were monitored using circular dichroism and fluorescence spectroscopy. The Cm values for GBNTD, GBCTD and GBF proteins were found to be 2.25, 1.65 and 1.82M during GdmCl-induced denaturation and 2.95, 2.25 and 2.67M for urea-induced denaturation. It is observed that GBNTD is more stable than GBCTD and it contributes to overall stability of GyrB. The lower Cm and ΔG values reflect the flexibility of GBCTD to form the catalytic site along with GANTD for cleavage or religation reaction. Both GdmCl- and urea-induced denaturation of GyrB domains were reversible over the entire range of concentration.