[en] The Bacillus licheniformis 74911 BlaI repressor is a prokaryotic regulator that, in the absence of a P-lactam antibiotic, prevents the transcription of the blaP gene, which encodes the BlaP beta-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-1-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 cooperative interactions between them. During the first step, the unfolding of the Blal CTD occurs, followed in the second step by the formation of an 'ANS-bound' intermediate state. Crosslinking 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 Blal NTD occurs at a GdmCI concentration of approx. 4 M. In summary, it is shown that the Blal CTD is structured, more flexible and less stable than the NTD upon GdmCI denaturation. These results contribute to the characterization of the Blal dimerization domain (i.e. CTD) involved in the induction process.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Vreuls, Christelle ; Université de Liège - ULiège > Département des sciences de la vie > Biologie et génétique moléculaire
Filée, Patrice ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Van Melckebeke, H.
Aerts, T.
De Deyn, P.
Llabres, Gabriel ; Université de Liège - ULiège > Département de physique > Département de physique
Matagne, André ; Université de Liège - ULiège > Département des sciences de la vie > Enzymologie
Simorre, J. P.
Frère, Jean-Marie ; Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Joris, Bernard ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Language :
English
Title :
Guanidinium chloride denaturation of the dimeric Bacillus licheniformis Blal repressor highlights an independent domain unfolding pathway
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