9 research outputs found
Structural contents of TtgR <sup>WT</sup> and mutants (%), in the absence of effectors.
<p>Data were obtained by deconvolution of the far UV CD spectra at 30°C.</p
Thermal denaturation followed by DSC of TtgR<sup>WT</sup> and variants (TtgR<sup>H114A</sup>, TtgR<sup>N110A</sup>, TtgR<sup>E78A</sup> and TtgR<sup>S77A</sup>) 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<sup>WT</sup> and variants (TtgR<sup>H114A</sup>, TtgR<sup>N110A</sup>, TtgR<sup>E78A</sup> and TtgR<sup>S77A</sup>) 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
Effect of naringenin and phloretin on the dissociation of TtgR<sup>WT</sup> and variants (TtgR<sup>H114A</sup>, TtgR<sup>N110A</sup> and TtgR<sup>E78A</sup>) from <i>ttgR-ttgABC</i> intergenic region by EMSA.
<p>(-) 1nM of free labeled operator DNA, (+) DNA-complex with 2 μM of TtgR<sup>WT</sup> and its variants, and DNA complex in the presence of 5μM of naringenin (Nar) and phloretin (Phlr).</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
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
Far UV CD experiments of TtgR<sup>WT</sup> and variants in the absence of effectors.
<p>(A) Far UV CD spectra at 30°C. (B) Thermal unfolding of TtgR<sup>WT</sup> and mutants monitored by CD ellipticity at 222 nm. Variants are represented in grey line (TtgR<sup>WT</sup>), black solid line (TtgR<sup>H114A</sup>), black dashed line (TtgR<sup>N110A</sup>), black dotted line (TtgR<sup>E78A</sup>) and black squares and line (TtgR<sup>S77A</sup>).</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
ITC isotherms for the binding of TtgR<sup>E78A</sup> 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<sup>WT</sup> and variants (TtgR<sup>H114A</sup>, TtgR<sup>N110A</sup>, TtgR<sup>E78A</sup> and TtgR<sup>S77A</sup>) 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