Structural analysis of the operator binding domain of Tn10-encoded

May 31, 1991 - putative -helix-tum-a-helix motif of the operator binding domain, was studied by ... mutations with reduced operator binding affinity (...
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Biochemistry 1992, 31, 1951-1960 H. F. (1985) Science 229, 941-945. Parker, C. A. (1968) Photoluminescence of Solutions, p 222, American Elsevier, Amsterdam. Pawagi, A. B., & Deber, C. M. (1990) Biochemistry 29, 950-955. Pinkofsky, H. B., Rampal, A. L., Cowden, M. A., & Jung, C. Y. (1978) J . Biol. Chem. 253, 4930-4937. Rampal, A. L., & Jung, C. Y. (1987) Biochim. Biophys. Acta 896, 287-294. Rampal, A. L., Jung, E. K. Y., Chin, J. J., Deziel, M. R., Pinkofsky, H. B., & Jung, C. Y. (1986) Biochim. Biophys. Acta 859, 135-142. Shanahan, M. F., Morris, D. P., & Edwards, B. M. (1987) J . Biol. Chem. 262. 5978-5984.

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Structural Analysis of the Operator Binding Domain of Tn 10-Encoded Tet Repressor: A Time-Resolved Fluorescence and Anisotropy Study Marie Chabbert,* Wolfgang Hillen,$ Dieter Hansen,siI1 Masayuki Takahashi,l and Jean-Alain Bousquet*l* CNRS UA 491, Laboratoire de Physique, Facultt de Pharmacie de Strasbourg, BP 24, F-67401 Nlkirch, France, Groupe de Canctrogdnhe et de Mutagdn5se Moldculaire et Structurale, IBMC du CNRS et Universitd Louis Pasteur, 15 rue Rend Descartes, F-67084 Strasbourg, France, and Lehrstuhl fur Mikrobiologie, Inrtitut fur Mikrobiologie und Biochemie der Friedrich- Alexander Universitat, Erlangen-Niirnberg, Staudtstrasse 5, G-8520 Erlangen, Federal Republic of Germany Received May 31, 1991; Revised Manuscript Received November 22, 1991

ABSTRACT: An engineered TnlO-encoded Tet repressor, bearing a single Trp residue at position 43, in the putative a-helix-tum-a-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.

R e g u l a t i o n of gene expression in procaryotes is often mediated by proteins which recognize DNA sequence specifically (Pabo & Sauer, 1984). The ability of many regulatory proteins to recognize the respective target DNA depends on the presence, e.g., Trp repressor (Gunsalus & Yanofsky, 1980), or the absence of effector molecules, e.g., Lac repressor (Miller & Reznikoff, 1980). X-ray analyses of the crystal structure of several repressors provide keys to the molecular basis of sequence specificity in DNA binding proteins. These data reveal that an a-helix-turn-a-helix structural motif is a common feature for sequencespecific DNA recognition among

* FacultC de Pharmacie de Strasbourg.

5 Institut fur

Mikrobiologie und Biochemie der Friedrich-Alexander Universitat. 11 Present address: Henkel KGaA, TFB Biotechnologie, Henkelst. 67, G-4000 Dusseldorf, FRG. IMBC du CNRS et Universite Louis Pasteur.

procaryotic DNA binding proteins (Pabo & Sauer, 1984). This a-helix-turn-a-helix motif makes sequence-specific contacts in the major groove of B-DNA. In this model, sequence specificity is achieved by interactions of amino acid side chains with solvent-exposed functions of the base pairs (Pabo & Sauer, 1984; Harrison & Aggarval, 1990). Tet repressor controls transcription of the Tn 10-encoded ret genes conferring resistance to tetracycline in Gram-negative bacteria (Beck et al., 1982; Bertrand et al., 1983; Hillen et al., 1984). This control is negatively regulated by the antibiotic tetracycline (Yang et al., 1976), which functions as an inducer (Hillen et al., 1983, 1984). Tet repressor has been purified and characterized (Hillen et al., 1982; Oehmichen et al., 1984) and its sequence has been determined (Postle et al., 1984). The comparison of Tet repressor with other repressor sequences (Pabo & Sauer, 1984) and the positions of trans-dominant mutations with reduced operator binding affinity (Isackson

0006-2960/92/0431-195 1$03.00/0 0 1992 American Chemical Society

1952 Biochemistry, Vol. 31, No. 7, 1992 & Bertrand, 1985) strongly suggest that an a-helix-turn-ahelix motif is involved in sequence-specific DNA binding. Comparison of the repressor gene sequences from each of the five classes of tetracycline resistance genes from Gramnegative bacteria (Mendez et al., 1980; Tovar et al., 1988) shows that the repressor protein from each class contains only two tryptophan residues at conserved positions (Unger et al., 1984). This suggests that these residues are important for repressor structure and/or function. In TnlO-encoded Tet repressor, these residues are located at position 43 and 75. Tet repressor mutants with either one or both of the tryptophans replaced by a phenylalanine have been engineered (Hansen & Hillen, 1987). These mutated proteins maintain the ability to bind ret operator DNA and tetracycline, albeit with altered affinity, and to act as a repressor (Hansen & Hillen, 1987; Hansen et al., 1987). A sequence-specific contact between Trp-43 and tet operator DNA has been suggested by fluorescence spectroscopy of these mutants and quantitative binding analysis of the respective complexes (Hansen & Hillen, 1987). As a matter of fact, Trp-43 is located in a-helix 3 of Tet repressor, part of the proposed a-helix-turn-a-helix motif. In this report, we summarize the results of a fluorescence analysis of F75 TetR, an engineered Tet repressor containing a single Trp residue at position 43 (Hansen & Hillen, 1987). Our data are consistent with the existence of an equilibrium between two classes of Trp-43. These classes are defined by different lifetimes, maximum emission wavelengths, correlation rotational times, and quencher accessibilities. These parameters are altered by the presence of disruptive agents and might provide information on the conformation of the a-helixturn-a-helix motif. AND METHODS MATERIALS Chemicals. All chemicals were of reagent grade or better. High-purity sodium chloride was purchased from Prolab, Tris was from Serva. Potassium iodide was from Merck and guanidine hydrochloridefrom Sigma. Ultrapure water (MilliQ instrument from Millipore Corp.) was used throughout the experiments. The dimer protein concentration usually ranged around 3 pM. All experiments were performed in 10 mM Tris-HC1, pH 8.0, 100 mM NaC1, 5 mM MgC12, and either 1 mM DTT or 10 mM 8-mercaptoethanol. The addition of other chemicals in the buffer will be noted specifically. The pH was adjusted to 8.00 f 0.05, after correction for the temperature dependence of the pK, of the Tris buffer. The temperature was controlled at 20 f 1 OC. F75 TetR Preparation. The mutated F75 Tet repressor differs from the wild type by the replacement of Trp-75 by Phe. After the preparation carried out as described in detail elsewhere (Hansen & Hillen, 1987), a further purification step using the FPLC system from Pharmacia was added to remove the fluorescence contamination previously observed (Hansen & Hillen, 1987). The sample was loaded on a MonoQ column equilibrated in 10 mM Tris-HC1, pH 8.0, and 0.1 mM DTT and eluted with a NaCl linear gradient from 10 mM Tris-HC1, pH 8.0, and 0.1 mM DTT to 10 mM Tris-HC1, pH 8.0, 0.1 mM DTT, and 0.5 M NaC1. The main peak, which eluted at 0.35 M NaC1, was collected and dialyzed against 0.1 M NaC1. It was free from fluorescent contamination, as controlled by using the double-mutant repressor, F43F75 TetR, whose two tryptophan residues were changed to Phe. The protein was precipitated by addition of ammonium sulfate until 60% saturation was reached and stocked at 4 OC in 10 mM Tris-HC1, pH 8.0, 100 mM NaC1,2 mM DTT, 1 mM EDTA, and 60% ammonium sulfate. Before measure-

Chabbert et al. ments, the protein solution was extensively dialyzed against 10 mM Tris-HC1, pH 8.0, 100 mM NaCl, and either 1 mM DTT or 10 mM j3-mercaptoethanol. The concentration of F75 TetR was determined by absorption, using an extinction coefficient of 7000 at 280 nm. This value was determined by measuring the absorption of a protein solution in 10 mM ammonium carbonate and, then, the concentration of the same solution by amino acid analysis. Spectroscopic Methods. UV absorption spectra were recorded on a Cary 219 spectrophotometer. Steady-state fluorescence measurements were performed with a MPF 66 spectrofluorometer (Perkin-Elmer). The excitation wavelength was set at 295 nm to avoid contribution from tyrosine residues. The quantum yields were determined relative to that of tryptophan zwitterion at pH 7.0 (0.14). The iodide quenching measurements were carried out by adding aliquots of a concentrated stock solution of iodide (6 M) to the protein solution. For each iodide concentration, the total fluorescence intensity was determined from the area under the spectrum and was corrected for the buffer baseline, the dilution effect, and the ionic strength effect. Fluorescence lifetime measurements were performed with the pulse fluorometry technique. A complete description of the device has been reported elsewhere (Montoro et al., 1988). Briefly, a mode-locked Argon laser (SpectraPhysics) synchronously pumped a rhodamine 6G dye laser (SpectraPhysics). The pulse repetition rate of the dye laser was controlled by a cavity dumper and set at 80 kHz. Output pulses (full width at half maximum 20 ps), tuned at 590 nm, were upconverted with a frequency doubler employing a temperature-tuned KDP crystal. The emission was detected by using the single-photon counting method with a Phillips XP 2020 photomultiplier, at a right angle to the excitation beam, through a polarizer set at 54.7O from the excitation beam polarization to eliminate artifacts due to the rotation of the protein molecule. The emission wavelength was selected with a 8-nm monochromator (Jobin-Yvon H10). A Schott WG 320 filter was added in front of the monochromator to eliminate most of the light scattering. To take into account the wavelength dependence of the photomultiplier, the instrument response functions (full width at half maximum around 500 ps) were determined by collecting the emission from a p-terphenyl solution in a cyclohexane-CC1, mixture (4:3) at the same wavelength as the decay studied. The fluorescence lifetime of p-terphenyl in this solution was < 20 ps, and, thus, its observed decay was indistinguishable from the response function (Kolber & Barkley, 1986). The data were recorded on a multichannel analyzer (Ortec 7100). For both the sample decay and the response function, the couning was stopped when the peak channel reached lo4 counts. The calibration was 108 ps/channel. The decay data were transmitted to an IBM 3090 computer and analyzed as sums of exponentials: I(t,A)

= &(A)e-r/Ti(A) i

(1)

with CCYi(A) = 1 i

by an iterative reconvolution procedure based on the Marquardt algorithm. The number of exponentials was progressively increased until the fit did not improve. The adequacy of the fit was judged by the value of reduced xz and by visual inspection of the autocorrelation functions and of the weighted residuals, defined as the difference between the calculated and

Fluorescence Study of the Operator Binding Domain of TetR

Biochemistry, Vol. 31, No. 7, 1992

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Table I: Analysis of Typical Fluorescence Decays of F75 TetR Using an Increasing Number of Exponential Components" (nm) n r 1 (ns) F, r 2 (ns) F2 r 3 (ns) F3 xz 320

1 3.3 1 49 2 4.4 0.82 0.9 0.18 3.3 3 4.6 0.79 1.1 0.18 0.07 0.03 1.6 360 1 4.2 1 17 2 4.9 0.90 1.3 0.10 1.6 3 4.9 0.90 1.3 0.10 0.09