Structural analysis of the operator binding domain of Tn10-encoded Tet repressor: a time-resolved fluorescence and anisotropy study

Abstract

An engineered Tn10-encoded Tet repressor, bearing a single Trp residue at position 43, in the putative alpha-helix-turn-alpha-helix motif of the operator binding domain, was studied by time-resolved fluorescence and anisotropy. Fluorescence intensity decay data suggested the existence of two classes of Trp-43, defined by different lifetimes. Analysis of anisotropy data were consistent with a model in which each class was defined by a different lifetime, rotational correlation time, and fluorescence emission maximum. The long-lifetime class had a red-shifted spectrum, similar to that of tryptophan zwitterion in water, and a short rotational correlation time. In contrast, the spectrum of the short-lifetime class was blue-shifted 10 nm compared to that of the long-lifetime class. Its correlation time was similar to that of the protein, which showed that Trp in this class was entirely constrained. Trp in this latter class could not be quenched by iodide, whereas most of the long-lifetime class was easily accessible. Presence of disruptive agents, such as 1 M GuCl or 3 M KCl, did not alter markedly the lifetimes but increased the weight of the short-lifetime component. In the same time, the rotational correlation time of this component was dramatically reduced. Taken together, our data suggest that the long-lifetime class could correspond to the tryptophan residues exposed to solvent whereas the short-lifetime class would correspond to the tryptophan residues embedded inside the hydrophobic core holding the helix-turn-helix motif. Destabilization of hydrophobic interactions would lead to an increase in the weight of the latter class for entropic reasons. Analysis of the fluorescence parameters of Trp-43 could provide structural information on the operator binding domain of Tet repressor.

A fluorescence study of Tn10-encoded Tet repressor

“…The downward curvature of the Stern-Volmer plots indicates that iodide is able to selectively quench the fluorescence emission of the fluorescing components which are more accessible to the solute. Our observation is in agreement with the other data reported on Tet repressor Chabbert et at., 1992), which show that downward curvature is observed for the quencher iodide. Equation (1) was fitted to our iodide quenching data by a nonlinear least-squares method (Stryjewski and Wasylewski, 1986).…”

“…The decay of the tryptophan residue in W43 TetR at 20~ is dominated by the r~ = 5.53 nsec component, which contributes about 91% of the emission, and the remaining 9% of the total emission corresponds to the shorter lifetime component characterized by r2 =2.36nsec. These values of fluorescence lifetime of W43 obtained by the frequency-domain method are slightly higher than the values of ~'l=4.9nsec, f~=0.90, and r2 = 1.3 nsec obtained by Chabbert et al (1992), who used the time-domain method.…”

“…We note that performed preliminary studies involving steady-state fluorescence measurements on wild type TetR as well as on two singletryptophan-containing mutants W43 and W75. More recently Chabbert et al (1992), using time-domain methods, described the time-resolved fluorecence emission of mutant TetR containing a single tryptophan residue at position 43.…”

“…Time-resolved fluorescence measurements have been used in order to study a mutant of Tnl0-encoded Tet repressor which possesses only one tryptophan residue at position 43 in the helix-turn-helix structure (Chabbert et al, 1992). The fluorescence decay and anisotropy analyses indicate the existence of two classes of W43 in Tet repressor.…”

“…The emission of the long-lifetime class is red-shifted as compared to the short-lifetime class. The tryptophan residue of the long-lifetime class can be quenched by iodide and is characterized by a short rotational correlation time (Chabbert et al, 1992). The authors suggest that the short-lifetime class could correspond to the tryptophan residue embedded inside the hydrophobic core holding the HTH motif in opposition to the long-lifetime class, which corresponds to the tryptophan residue exposed to solvent.…”