A differential scanning calorimetry study of tetracycline repressor.
Tetracycline repressor (TetR), which constitutes the most common mechanism of bacterial resistance to an antibiotic, is a homodimeric protein composed of two identical subunits, each of which contains a domain possessing a helix-turn-helix motif and a domain responsible for binding tetracycline. Binding of tetracycline in the protein pocket is accompanied by conformational changes in TetR, which abolish the specific interaction between the protein and DNA. Differential scanning calorimetry (DSC) and CD measurements, performed at pH 8.0, were used to observe the thermal denaturation of TetR in the absence and presence of tetracycline. The DSC results show that, in the absence of tetracycline, the thermally induced transitions of TetR can be described as an irreversible process, strongly dependent on scan rate and indicating that the protein denaturation is under kinetic control described by the simple kinetic scheme: N(2)--->D(2), where k is a first-order kinetic constant, N is the native state, and D is the denatured state. On the other hand, analysis of the scan rate effect on the transitions of TetR in the presence of tetracycline shows that thermal unfolding of the protein can be described by the two-state model: N(2)<--->U(2)--->D. In the proposed model, TetR in the presence of tetracycline undergoes co-operative unfolding, characterized by an enthalpy change (DeltaH(cal) = 1067 kJ x mol(-1)) and an entropy change (DeltaS = 3.1 kJ x mol(-1)).
Kedracka-Krok
Sylwia
S
Department of Physical Biochemistry, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland.
Wasylewski
Zygmunt
Z
D002152:Calorimetry, Differential Scanning / Q000379:methods*; D002942:Circular Dichroism; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry; D007700:Kinetics; D012097:Repressor Proteins / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein