Previous studies [Wasylewski et al. (1996), J. Protein Chem. 15, 45-58] have shown that the W43 residue localized within the helix-turn-helix structure domain of Tet repressor can exist in the ground state in two conformational states. In this paper we investigate the fluorescence properties of W43 of TetR upon binding of tetracycline inducer and its chemical analogs such as anhydro- and epitetracycline. Binding of the drug inducer to the protein indicates that the W43 residue still exists in two conformational states; however, its environment changes drastically, as can be judged by the changes in fluorescence parameters. The FQRS (fluorescence-quenching-resolved spectra) method was used to decompose the total emission spectrum. The resolved spectra exhibit maxima of fluorescence at 346 and 332 nm and the component quenchable by KI (346 nm) is shifted 9 nm toward the blue side of the spectrum upon inducer binding. The observed shift does not result from the changes in the exposure of W43, since the bimolecular quenching rate constant remains the same and is equal to about 2.7 x 10(9) M-1 sec-1. The binding of tetracycline leads to drastic decrease of the W43 fluorescence intensity and increase of the tetracycline intensity as well as the decrease of fluorescence lifetime, especially of the W43 component characterized by the emission at 332 nm. The observed energy transfer from W43 to tetracycline is more efficient for the state characterized by the fluorescence emission at 332 nm (88%) than for the component quenchable by iodide (53%). Tetracycline and several of its derivatives were also used to observe how chemical modifications of the hydrophilic groups in tetracycline influence the mechanism of binding of the antibiotic to Tet repressor. By use of pulsed-laser photoacoustic spectroscopy it is shown that the binding of tetracyclines to Tet repressor leads to significant increase of tetracycline fluorescence quantum yields. Steady-state fluorescence quenching of tetracycline analogs in complexes with Tet repressor using potassium iodide as a quencher allowed us to determine the dependence of the exposure of bound antibiotic on the modifications of hydrophilic substituents of tetracycline. Circular dichroism studies of the TetR-[Mg.tc]+ complex do not indicate dramatic changes in the secondary structure of the protein; however, the observed small decrease in the TetR helicity may occur due to partial unfolding of the DNA recognition helix of the protein. The observed changes may play an important role in the process of induction in which tetracycline binding results in the loss of specific DNA binding.
Steady-state fluorescence quenching and time-resolved measurements have been performed to resolve the fluorescence contributions of the two tryptophan residues, W43 and W75, in the subunit of the homodimer of the Tet repressor from Escherichia coli. The W43 residue is localized within the helix-turn-helix structural domain, which is responsible for sequence-specific binding of the Tet repressor to the tet operator. The W75 residue is in the protein matrix near the tetracycline-binding site. The assignment of the two residues has been confirmed by use of single-tryptophan mutants carrying either W43 or W75. The FQRS (fluorescence-quenching-resolved-spectra) method has been used to decompose the total emission spectrum of the wild-type protein into spectral components. The resolved spectra have maxima of fluorescence at 349 and 324 nm for the W43 and W75 residues, respectively. The maxima of the resolved spectra are in excellent agreement with those found using single-tryptophan-containing mutants. The fluorescence decay properties of the wild type as well as of both mutants of Tet repressor have been characterized by carrying out a multitemperature study. The decays of the wild-type Tet repressor and W43-containing mutant can be described as being of double-exponential type. The W75 mutant decay can be described by a Gaussian continuous distribution centered at 5.0 nsec with a bandwidth equal to 1.34 nsec. The quenching experiments have shown the presence of two classes of W43 emission. One of the components, exposed to solvent, has a maximum of fluorescence emission at 355 nm, with the second one at about 334 nm. The red-emitting component can be characterized by bimolecular-quenching rate constant, kq equal to 2.6 x 10(9), 2.8 x 10(9), and 2.0 x 10(9) M-1 sec-1 for acrylamide, iodide, and succinimide, respectively. The bluer component is unquenchable by any of the quenchers used. The W75 residue of the Tet repressor has quenching rate constant equal to 0.85 x 10(9) and 0.28 x 10(9) M-1 sec-1 for acrylamide and succinimide, respectively. These values indicate that the W75 is not deeply buried within the protein matrix. Our results indicate that the Tet repressor can exist in its ground state in two distinct conformational states which differ in the microenvironment of the W43 residue.
Effects of low‐energy implantation on the properties of subsurface layers of high speed tool steel are investigated. The depth profiles of nitrogen and titanium are measured. The results are discussed in terms of adhesion of hard thin film (TiN) to the implanted surface.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.