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.

The time dependence of the human alpha 1-antitrypsin polymerization process was studied by means of the intrinsic fluorescence stopped-flow technique as well as the fluorescence-quenching-resolved spectra (FQRS) method and native PAGE. The polymerization was induced by mild denaturing conditions (1 M GuHCl) and temperature. The data show that the dimer formation reaction under mild conditions was followed by an increase of fluorescence intensity. This phenomenon is highly temperature sensitive. The structure of alpha 1-antitrypsin dimer resembles the conformation of antithrombin III dimer. In the presence of the denaturant the polymerization process is mainly limited to the dimer state. The alpha 1-antitrypsin activity measurements confirm monomer-to-dimer transition under these conditions. These results are in contrast to the polymerization process induced by temperature, where the dimer state is an intermediate step leading to long-chain polymers. On the basis of stopped-flow and electrophoretic data it is suggested that both C-sheet as well as A-sheet mechanisms contribute to the polymerization process under mild conditions.

The application of a new fluorescence-quenching-resolved spectroscopic method [Wasylewski, Z., Koloczek, H. and Wahiowska, A. (1988) Eur. J . Biochem. 172, 719-7241 for resolving fluorescence emission spectra of a mixture of fluorophores into components is described. Contrary to fluorescence lifetime measurements, in this method the overlapping spectra can be decomposed even if the components have similar or the same fluorescence lifetimes, but differ in bimolecular-rate-quenching constants. Using this technique, we have resolved the emission spectra of a two-component mixture of fluorescein and riboflavin, which have very similar fluorescence lifetimes. To illustrate the utility of this approach in the study of fluorophores in compartmentalized biological systems such as lipid bilayers, we have also used the method to resolve the emission spectra of a two-component mixture of fluorophores commonly used in biological studies which undergo partition between water and a micellar phase.Fluorescence spectroscopy is frequently used as a quantitative method to analyze complex systems in the physical, chemical and biological sciences. The resolution of fluorescence emission heterogeneity, which can result both from ground-state heterogeneity and excited-state reactions [l], provides more detailed information about the mixture of fluorophore components or information about the environment of the fluorophore probes [l]. In order to resolve the fluorescence emission spectra of a heterogeneous system of a mixture of aromatic fluorophores or of two tryptophan residues in proteins, the time-domain [l -61 and phase-modulation techniques [7 -111 have been used. Both these approaches require the fluorescence lifetimes of the components investigated to be significantly different. To resolve the overlapping spectra one has to obtain a number of decay measurements at different emission wavelengths.Recently [12], we have developed a powerful new method which involves the determination of fluorescence-quenchingresolved spectra (FQRS). In this new procedure steady-state emission spectra are collected at several fluorescence quencher concentrations. Then the Stern-Volmer quenching analysis, performed by an iterative non-linear least-squares method at each particular wavelength, allows resolution of the emission spectra into components.In the FQRS method, the possibility of resolving the fluorescence emission spectra depends on differences in the dynamic Stern-Volmer quenching constants, K , of the components. Since the Stern-Volmer constant for an efficient quencher is proportional both to the fluorescence lifetime T~ and bimolecular rate quenching constant k,, a mixture of fluorophores differing in T~ and/or k, can be successfully resolved into components.We report here the first FQRS measurements for a twocomponent mixture of fluorophores of biological interest, which have similar fluorescence lifetimes, such as riboflavin and fluorescein. The FQRS method has also been used to resolve the heterogeneous fluorescence of probes (diph...

Cyclic AMP receptor protein (CRP) regulates the expression of more than 100 genes in Escherichia coli when complexed with cyclic AMP. Dynamic light scattering (DLS) and fluorescence decay anisotropy measurements of CRP were performed in solution, in the absence and presence of cAMP. We have also measured the effect of DNA sequences, including lac and gal promoter sequences, on the shape of CRP-DNA complexes. DLS measurements show that upon cAMP binding at low nucleotide concentration, the Stokes radius decreases from the value of 2.8 nm for apo-CRP to the value of 2.7 nm. At higher cAMP concentration, only a very small further decrease was detected. Fluorescence anisotropy decay measurements, with the use of CRP labeled at Cys-178 with 1,5-I-AENS, indicate that apo-CRP exhibits two rotational correlation times. The longer time, theta1 = 23.3 ns, corresponds to the overall motion of the protein, and the shorter time, theta2 = 1.4 ns, exhibits segmental mobility of the C-terminal domain of CRP. Binding of cAMP into CRP induced substantial increase of theta1 to the value of 30.7 ns, whereas theta2 remained unchanged. The DLS measurements indicate that the binding of CRP into a fragment of DNA possessing a sequence of lac promoter induces a larger increase in the Stokes radius of lac-CRP complex than in case of gal-CRP complex. Similarly, a higher change was detected in rotational correlation time, theta1, in the case of lac-CRP complex than in case of gal-CRP. Because the lac and gal promoters are characteristic for the two different classes of CRP-dependent promoters, one can expect that the observed differences in lac-CRP and gal-CRP complexes are important in activation of transcription in Escherichia coli.

The dependence of fluorescence emission maxima of L-tryptophan and single-tryptophan-containing proteins (ribonuclease T1, melittin, and parvalbumin) on excitation wavelength has been studied in reversed micelle systems of sodium bis(2-ethyl-1-oxyl) sulfosuccinate (AOT). No effect of fluorescence maximum shift for different excitation wavelengths is observed for ribonuclease T1, in which a single tryptophan residue is located in the nonrelaxating, nonpolar protein interior. L-Tryptophan and the rest of the studied proteins, which contain single tryptophan residues exposed to the solvent, exhibit the dipolar relaxational processes of partly immobilized water molecules in micelles. This effect depends on the molar H2O/AOT ratio. Circular dichroism measurements prove that there have been no structural changes of the studied proteins in micellar systems. The results provide information about dynamic relaxational processes in proteins.