Conformational stability and ligand binding properties of BldR, a member of the MarR family, from Sulfolobus solfataricus.
The multiple antibiotic resistance regulators (MarR) constitute a family of ligand-responsive transcriptional regulators ubiquitous among the bacterial and archaeal domains. BldR, an archaeal MarR member characterized from the hyperthermophilic crenarchaeon Sulfolobus solfataricus regulates its own expression and that of an alcohol dehydrogenase gene by binding to sequences in their promoters and responding to benzaldehyde as the effector molecule. In this study we assessed the thermodynamic stability of the protein BldR and its binding with benzaldehyde through biophysical measurements. The temperature- and denaturant-induced unfolding experiments, performed by means of circular dichroism (CD) and differential scanning calorimetry (DSC), showed that BldR has an extremely high thermal stability (Td=108.9°C) and a remarkable resistance against GuHCl (Cm=5.3M at 25°C). The unfolding Gibbs energy, ΔdG (H2O), calculated by the linear extrapolation model from GuHCl-induced unfolding equilibrium curve, is 72.2kJmol(-1). ITC binding experiments showed that four benzaldehyde molecules bind to one BldR dimer with a binding constant Kb of 7.5·10(5)M(-1), being the binding entropically driven. ITC, CD and fluorescence results are consistent with a conformational change induced by benzaldehyde binding, further proving that this molecule is a specific effector for BldR modulating its DNA binding activity.
Department of Biology, University of Naples Federico II, via Cintia, ed 7, 80126 Naples, Italy. Electronic address: gabriella.fiorentino@unina.it.
Gabriella
G
Fiorentino
Department of Biology, University of Naples Federico II, via Cintia, ed 7, 80126 Naples, Italy.
Immacolata
I
Del Giudice
Department of Chemical Sciences, University of Naples Federico II, via Cintia, 80126 Naples, Italy.
Luigi
L
Petraccone
Department of Biology, University of Naples Federico II, via Cintia, ed 7, 80126 Naples, Italy.
Simonetta
S
Bartolucci
Department of Chemical Sciences, University of Naples Federico II, via Cintia, 80126 Naples, Italy. Electronic address: pompea.delvecchio@unina.it.
Pompea
P
Del Vecchio
D019843:Archaeal Proteins / Q000737:chemistry*; D001547:Benzaldehydes / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry; D048229:Sulfolobus solfataricus / Q000737:chemistry*; D013816:Thermodynamics
D019843:Archaeal Proteins; D001547:Benzaldehydes; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D019791:Guanidine; C032175:benzaldehyde
Archaea; Benzaldehyde binding; DSC; Hyperthermophile; ITC; Thermodynamic stability; Winged-helix transcriptional regulator