NMR studies on the equilibrium unfolding of ketosteroid isomerase by urea

Abstract

Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of H-1-N-15 HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the beta 5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the beta 6-strand. The results thus suggest that the interface region primarily around the beta 5- and beta 6-strands could play an important role as the starting positions in the unfolding process of KSI.