Structural analysis of the operator binding domain of Tn10-encoded Tet repressor: a time-resolved fluorescence and anisotropy study.
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.
Chabbert
M
M
CNRS UA 491, Faculté de Pharmacie de Strasbourg, Illkirch, France.
Hillen
W
W
Hansen
D
D
Takahashi
M
M
Bousquet
J A
JA
D004251:DNA Transposable Elements; D005454:Fluorescence Polarization; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D009875:Operator Regions, Genetic; D011189:Potassium Chloride / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D011815:R Factors / Q000235:genetics*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics*
D004251:DNA Transposable Elements; D006146:Guanidines; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D011189:Potassium Chloride; D019791:Guanidine