Estimation of structure and stability of MurE ligase from Salmonella enterica serovar Typhi

MurE ligase catalyzes the assembly of peptide moiety, an essential component of bacterial cell wall. We have explored the conformational stability and unfolding equilibrium behaviour of the protein MurE ligase by determining the conformational free energy, entropy and enthalpy parameters under stress conditions. MurE from Salmonella enterica Serovar Typhi was cloned, expressed and purified. Conformational changes associated with increasing concentration of GdmCl- and urea-induced denaturation of MurE were monitored using Circular Dichroism (CD) and fluorescence spectroscopies. The secondary structural content of protein estimated by CD experiment is in close agreement with the predicted MurE ligase structure by homology modeling. Denaturant-induced transition curve was analyzed for thermodynamic parameters. Average values for MurE ligase of ΔG_D⁰ = 3.13 kcal mol^-1, m = 1.52 kcal mol^-1 M^-1 and C_m (=ΔG_D⁰/m) = 2.05 M were calculated in the presence of GdmCl whereas in the case of urea these were ΔG_D⁰ = 3.04 kcal mol^-1, m = 1.20 kcal mol^-1 M^-1 and C_m (=ΔG_D⁰/m) = 2.53 M. The observed superposition of normalized transition curve of two independent optical properties suggested that GdmCl- and urea-induced denaturation follow a two-state process.