Probing the equilibrium unfolding of ketosteroid isomerase through xenon-perturbed H-1-N-15 multidimensional NMR spectroscopy

Abstract

We used xenon-perturbed H-1-N-15 multidimensional NMR to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Three limited regions located on the beta 3-, beta 5- and beta 6-strands of dimeric interface were significantly perturbed by urea in the early stage of KSI unfolding, which could lead to dissociation of the dimer into structured monomers at higher denaturant concentration as the interactions in these regions are weakened. The results indicate that the use of xenon as an indirect probe for multidimensional NMR can be a useful method for the equilibrium unfolding study of protein at residue level.