Thermal denaturation followed by DSC of TtgR^WT and variants (TtgR^H114A, TtgR^N110A, TtgR^E78A and TtgR^S77A) in the presence and in the absence of ligands (black solid lines free; grey solid lines 250 μM of chloramphenicol; grey dashed lines 250 μM naringenin; grey dotted lines 250 μM phloretin).

<p>Thermal denaturation followed by DSC of TtgR^WT and variants (TtgR^H114A, TtgR^N110A, TtgR^E78A and TtgR^S77A) in the presence and in the absence of ligands (black solid lines free; grey solid lines 250 μM of chloramphenicol; grey dashed lines 250 μM naringenin; grey dotted lines 250 μM phloretin).</p

Structural contents of TtgR ^WT and mutants (%), in the absence of effectors.

<p>Data were obtained by deconvolution of the far UV CD spectra at 30°C.</p

Thermodynamic parameters of the association of TtgR and mutants with chloramphenicol, naringenin and phloretin.

<p>Thermodynamic parameters of the association of TtgR and mutants with chloramphenicol, naringenin and phloretin.</p

Theoretical energy measurements of models.

<p>*The values are presented in ΔΔG (kcal/mol) as the difference between the free energy of mutants with respect to the free energy of wild type. <b>Effector free</b>: stability energy calculated as the difference between folded and unfolded states. <b>With effectors</b>: free energy of binding calculated as the difference between the bound and unbound state.</p><p>Theoretical energy measurements of models.</p

Effect of naringenin and phloretin on the dissociation of TtgR^WT and variants (TtgR^H114A, TtgR^N110A and TtgR^E78A) from <i>ttgR-ttgABC</i> intergenic region by EMSA.

<p>(-) 1nM of free labeled operator DNA, (+) DNA-complex with 2 μM of TtgR^WT and its variants, and DNA complex in the presence of 5μM of naringenin (Nar) and phloretin (Phlr).</p

Far UV CD experiments of TtgR^WT and variants in the absence of effectors.

<p>(A) Far UV CD spectra at 30°C. (B) Thermal unfolding of TtgR^WT and mutants monitored by CD ellipticity at 222 nm. Variants are represented in grey line (TtgR^WT), black solid line (TtgR^H114A), black dashed line (TtgR^N110A), black dotted line (TtgR^E78A) and black squares and line (TtgR^S77A).</p

Sequence alignment of close homologues of TtgR (<i>Pseudomonas putida</i> DOT-T1E).

<p>Mutated residues (S77, E78, N110 and H114) are boxed and shaded.</p

ITC isotherms for the binding of TtgR^E78A mutant to: A) chloramphenicol, B) naringenin and C) phloretin.

<p>Experiments were performed as described in “Materials and Methods section” at 30°C with injections of the effector diluted in dialysis buffer into the protein. Upper panels: Heats changes for injections. Lower panels: Experimental heats measured for each effector injection. The curves correspond to the best fit using “one type of sites” model.</p

Thermodynamic parameters obtained by DSC and CD.

<p>Thermodynamic parameters for the thermal unfolding of TtgR^WT and variants (TtgR^H114A, TtgR^N110A, TtgR^E78A and TtgR^S77A) obtained from DSC and far-UV CD measurements. Internal calibrations of the CD and DSC equipments give errors in Tm of ± 0.20°C and ± 0.10°C respectively.</p