Guanidinium chloride denaturation of the dimeric Bacillus licheniformis BlaI repressor highlights an independent domain unfolding pathway

Abstract

The Bacillus licheniformis 749/I BlaI repressor is a prokaryotic regulator that, in the absence of a β-lactam antibiotic, prevents the transcription of the blaP gene, which encodes the BlaP β-lactamase. The BlaI repressor is composed of two structural domains. The 82-residue NTD (N-terminal domain) is a DNA-binding domain, and the CTD (C-terminal domain) containing the next 46 residues is a dimerization domain. Recent studies have shown the existence of the monomeric, dimeric and tetrameric forms of BlaI in solution. In the present study, we analyse the equilibrium unfolding of BlaI in the presence of GdmCl (guanidinium chloride) using different techniques: intrinsic and ANS (8-anilinonaphthalene-l-sulphonic acid) fluorescence, far- and near-UV CD spectroscopy, cross-linking, analytical ultracentrifugation, size exclusion chromatography and NMR spectroscopy. In addition, the intact NTD and CTD were purified after proteolysis of BlaI by papain, and their unfolding by GdmCl was also studied. GdmCl-induced equilibrium unfolding was shown to be fully reversible for BlaI and for the two isolated fragments. The results demonstrate that the NTD and CTD of BlaI fold/unfold independently in a four-step process, with no significant co-operative interactions between them. During the first step, the unfolding of the BlaI CTD occurs, followed in the second step by the formation of an ‘ANS-bound’ intermediate state. Cross-linking and analytical ultracentrifugation experiments suggest that the dissociation of the dimer into two partially unfolded monomers takes place in the third step. Finally, the unfolding of the BlaI NTD occurs at a GdmCl concentration of approx. 4 M. In summary, it is shown that the BlaI CTD is structured, more flexible and less stable than the NTD upon GdmCl denaturation. These results contribute to the characterization of the BlaI dimerization domain (i.e. CTD) involved in the induction process.