A differential scanning calorimetry study of tetracycline repressor

Abstract

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 helixturn-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 À! k 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 À! D. In the proposed model, TetR in the presence of tetracycline undergoes co-operative unfolding, characterized by an enthalpy change (DH cal ¼ 1067 kJAEmol )1 ) and an entropy change (DS ¼ 3.1 kJAEmol )1 ).

An Exclusive α/β Code Directs Allostery in TetR–Peptide Complexes

“…19,20,27 This is reflected by an increase in the midpoint of the denaturant concentration from 3.2 M to 4.8 M urea (Fig. S5) and by an increase in T m from 58°C to 71.0°C upon addition of ATc to TetR (Reichheld et al, 20 Kedracka-Krok and Wasylewski, 27 and this study). In order to investigate whether peptide binding leads to a similar stabilization of TetR, we performed urea-induced denaturation experiments monitored by CD spectroscopy (Fig.…”

Compartmentalized Glucose Metabolism in Pseudomonas putida Is Controlled by the PtxS Repressor

“…A number of transcriptional regulators consisting of an effector binding domain and a DNA binding domain have been described previously, i.e., TetR (2) and the NmrA regulator (16), to cite some, and have been analyzed by DSC. These studies showed that these proteins unfold in a single event, pointing toward the cooperative unfolding of both domains (14,16). In contrast, PtxS domains seem to unfold independently, as suggested by our DSC studies shown in Fig.…”

High Specificity in CheR Methyltransferase Function

“…DSC studies of other proteins have revealed in a number of cases that cooperative domain unfolding reflects functional interdomain cross-talk (57)(58)(59). Data thus suggest functional interdomain interaction in CheR.…”