Tài liệu Báo cáo khoa học: Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia coli ppt

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Steady-state and time-resolved fluorescence studies of conformationalchanges induced by cyclic AMP and DNA binding to cyclic AMPreceptor protein fromEscherichia coliAgnieszka Polit, Urszula Błaszczyk and Zygmunt WasylewskiDepartment of Physical Biochemistry, Faculty of Biotechnology, Jagiellonian University, Krako´w, PolandcAMP receptor protein (CRP), allosterically activated bycAMP, regulates the expression of several genes in Escheri-chia coli. As binding of cAMP leads to undefined conform-ational changes in CRP, we performed a steady-state andtime-resolved fluorescence study to show how the binding ofthe ligand influences the structure and dynamics of theprotein. We used CRP mutants containing a single trypto-phan residue at position 85 or 13, and fluorescently labeledwith 1,5-I-AEDANS attached to Cys178. Binding of cAMPin the CRP–(cAMP)2complex leads to changes in the Trp13microenvironment, whereas its binding in the CRP–(cAMP)4complex alters the surroundings of Trp85. Time-resolved anisotropy measurements indicated that cAMPbinding in the CRP–(cAMP)2complex led to a substantialincrease in the rotational mobility of the Trp13 residue.Measurement of fluorescence energy transfer (FRET)between labeled Cys178 and Trp85 showed that the bindingof cAMP in the CRP–(cAMP)2complex caused a substan-tial increase in FRET efficiency. This indicates a decrease inthe distance between the two domains of the protein from26.6 A˚in apo-CRP to 18.7 A˚in the CRP–(cAMP)2com-plex. The binding of cAMP in the CRP–(cAMP)4complexresulted in only a very small increase in FRET efficiency. Theaverage distance between the two domains in CRP–DNAcomplexes, possessing lac, gal or ICAP sequences, shows anincrease, as evidenced by the increase in the average distancebetween Cys178 and Trp85 to % 20 A˚. The spectral changesobserved provide new structural information about thecAMP-induced allosteric activation of the protein.Keywords: allosteric regulation; cAMP receptor protein;emission anisotropy; Escherichia coli; fluorescence.cAMP receptor protein (CRP), which is allostericallyactivated by cAMP, regulates transcription of over 100genes in Escherichia coli [1,2]. Upon binding the cyclicnucleotide, CRP undergoes an allosteric conformationalchange that allows it to bind specific DNA sequences withincreased affinity [3]. CRP is a dimeric protein, composed oftwo identical 209-amino-acid subunits. Each subunit ofCRP has a molecular mass of 23.6 kDa, as deduced fromthe amino-acid sequence. Individual subunits fold into twodomains [4]. The larger N-terminal domain (residues 1–133)is responsible for dimerization of CRP and for interactionwith the allosteric effector, cAMP. The smaller C-terminaldomain (residues 139–209) is responsible for interactionwith DNA through a helix–turn–helix motif. CRP recog-nizes a 22-bp, symmetric DNA site [5]. Amino-acid residues134–138 form a flexible hinge which covalently couples twodomains. Recent studies of the crystal structure of the CRP–DNA complex showed that each protein subunit binds twocAMP molecules with different affinities [6]. Higher-affinitysites, where the nucleotide binds in the anti conformation,are buried within the N-terminal domains, whereas lower-affinity binding sites (where the bound cAMP has a synconformation) are located at the interface formed by thetwo C-terminal domains of the CRP subunits, interactingwith a helix–turn–helix motif and, indirectly, with the DNA.Crystallographic observations have been supported byrecent NMR [7] and isothermal titration calorimetry studies[8]. Therefore, it has been suggested that CRP exists in threeconformational states: free CRP, CRP with two cAMPmolecules bound to N-terminal domains [CRP–(cAMP)2],and CRP with four cAMP molecules bound to bothN-terminal and C-terminal domains [CRP–(cAMP)4]. Anearlier hypothesis suggested [9] that the three conforma-tional states of CRP consisted of the following species: freeCRP, CRP–(cAMP)1and CRP–(cAMP)2, which has beenreinterpreted by Passner & Steitz [6]. It is important to notethat the behavior of CRP at different concentrations ofcAMP is essentially biphasic, so two different conformersexist at lower and higher concentrations of cAMP. In thepresence of % 100 lMcAMP, CRP becomes activated and isable to recognize and bind specific DNA sequences andstimulate transcription [10], whereas at millimolar concen-trations of cAMP, there is a loss of affinity and sequencespecificity for DNA binding and, consequently, loss oftranscription stimulation [11]. In the crystal phase, the CRPCorrespondence to Z.Wasylewski,DepartmentofPhysicalBiochemistry, Faculty of Biotechnology, Jagiellonian University,ul. Gronostajowa 7, 30-387 Krako´w, Poland.Fax: + 48 12 25 26 902, Tel.: + 48 12 25 26 122,E-mail: wasylewski@mol.uj.edu.plAbbreviations: 1,5-I-AEDANS, N-iodoacetylaminoethyl-1-naphthyl-amine-5-sulfonate; AEDANS-CRP, CRP covalently labeled with1,5-I-AEDANS attached to Cys178; apo-CRP, unligated CRP;CRP, cAMP receptor protein; FRET, fluorescence resonanceenergy transfer.(Received 31 October 2002, revised 19 December 2002,accepted 3 February 2003)Eur. J. Biochem. 270, 1413–1423 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03497.xdimer with two molecules of cAMP bound to anti-cAMPbinding sites is asymmetric, i.e. one monomer is in an ÔopenÕform, in which the a-helices are swung out away from theN-terminal domain, and the other monomer is in a ÔclosedÕform, in which the a-helices are swung in close to theN-terminal domain [4]. X-ray crystal structure studiesrevealed that the cAMP-ligated CRP dimer complexed toa 30-bp DNA sequence is exclusively in the ÔclosedÕ form [12].Each subunit of CRP contains two tryptophan residues atpositions 13 and 85 (Fig. 1). Both residues are located in theN-terminal domain, Trp85 near the cAMP-binding pocketand Trp13 on the surface of the protein. Trp13 is much moreaccessible to solvent than Trp85.19F-NMR studies haverevealed that binding of cAMP induces not only changes inthe immediate environment of the cAMP-binding site butalso far-reaching conformational changes (perturbation ofchemical shift of Trp13) [13]. Fluorescence studies haveshown that Trp13 is responsible for % 80% of the tryptophanfluorescence in CRP with % 20% of the signal originatingfrom Trp85 [14]. Acrylamide-mediated and iodide-mediatedfluorescence quenching studies indicate that Trp13 is solvent-exposed and accessible to the quenching agents. On the otherhand, Trp85 is inaccessible to quenching agents. Heyduk &Lee [9] have shown that, at micromolar cAMP concentra-tions, there is no detectable change in fluorescence intensityof protein tryptophan residues. The increase was onlydetected in the millimolar range of cAMP concentration.Biochemical and biophysical studies have demonstratedthat binding of cAMP allosterically induces CRP to assume aconformation that binds to DNA and interacts with RNApolymerase. However, the details of the mechanism by whichcAMP mediates the allosteric activation of CRP remainobscure, because the crystal structure of apo-CRP has not yetbeen elucidated. Therefore, we decided to study the changesin the apo-CRP structure induced by binding of cAMP andDNA by measuring fluorescence resonance energy transfer(FRET) and fluorescence anisotropy decay. Two tryptophanresidues, Trp13 and Trp85, were used as intrinsic donors forFRET analysis. The fluorescence anisotropy decay was usedto investigate the dynamics of CRP.Experimental proceduresMaterialsAcrylamide, KCl, EDTA, phenylmethanesulfonyl fluoride,Tris and N-iodoacetylaminoethyl-1-naphthylamine-5-sulfo-nate (1,5-I-AEDANS) were purchased from Sigma. cAMPand dithiothreitol were from Fluka. The Fractogel EMDSO3–650 (M) was from Merck, and Q-Sepharose Fast Flow,Sephacryl S-200 HR and Sephadex G-25 were fromAmersham Pharmacia Biotech. DNA sequence containingthe CRP-binding sites was from TIB MOLBIOL (Poznan´,Poland). The nutrients for bacterial growth were from LifeTechnologies. All other chemicals were analytical-gradeproducts of POCh-Gliwice (Gliwice, Poland). All measure-ments were performed in buffers prepared in water purifiedwith the Millipore system.The sequences of duplexes used in this study were asfollows:lac (26 bp), 5¢-ATTAATGTGAGTTAGCTCACTCATTA-3¢ and 3¢-TAATTACACTCAATCGAGTGAGTAAT-5¢;gal (26 bp), 5¢-AAAAGTGTGACATGGAATAAATTAGT-3¢ and 3¢-TTTTCACACTGTACCTTATTTAATCA-5¢;ICAP (28 bp), 5¢-AATTAATGTGACATATGTCACATTAATT-3¢ and 3¢-TTAATTACACTGTATACAGTGTAATTAA-5¢.The recognition half-sites are shown in bold. Anequimolar amount of complementary strand was added,and the mixture was heated for 1 min at 96 °C and slowlycooled to room temperature. The double-stranded DNAwas stored at )20 °C in experimental buffer.Protein purificationThe tryptophans at positions 13 and 85 of CRP were replacedwith phenylalanine and alanine, respectively. The mutagen-esis was performed using the overlap extension method withPwo DNA polymerase. pHA7 plasmid encoding mutant crpgenes was introduced into E. coli strain M182Dcrp, kindlyprovided by Dr S. Busby (The University of Birmingham,UK). The bacteria were grown on Luria–Bertani medium at37 °C overnight in a Biostat B fermentor from B. BraunBiotech International (Melsungen, Germany). Proteins werepurified at 4 °C, essentially as described previously [15] butwith one modification. After ion-exchange chromatographyon Q-Sepharose, the proteins were additionally purified bygel filtration on Sephacryl S-200 HR. After this procedure,the proteins were highly pure (> 97%), as judged by SDS/PAGE and Coomassie Brilliant Blue staining.For spectrophotometric determination of concentrations,the following absorption coefficients were used: 14 650M)1Æcm)1at 259 nm for cAMP [16] and 6000M)1Æcm)1atFig. 1. Structure of the CRP dimer. The locations of tryptophan resi-dues are marked in red, and the location of Cys178 residue is indicatedin yellow. The figure was generated withWEBLAB VIEWERPRO(version3.7) using atomic coordinates for the CRP–cAMP complex [29]. Thecoordinates were obtained from the Brookhaven Protein Data Bank(accession code 1G6N).1414 A. Polit et al.(Eur. J. Biochem. 270) Ó FEBS 2003340 nm for AEDANS [18]. Absorption coefficients of CRPmutants were determined using the method describedelsewhere [19] as 29 700M)1Æcm)1and 33 100M)1Æcm)1at278 nm for the W13F and W85A dimers, respectively.Measurements were performed in 50 mMTris/HClbuffer, pH 8.0, containing 100 mMKCl and 1 mMEDTA (buffer A), and 50 mMTris/HCl buffer, pH 7.8,supplemented with 100 mMKCl and 1 mMEDTA(buffer B).Fluorescence labeling of CRPCovalent modification of Trp mutants with 1,5-I-AEDANSwas carried out as described elsewhere [20] with severalmodifications. The protein and label were mixed at a molarratio of 1 : 10 and incubated at room temperature for 2 h,andthenat4°C overnight in the dark. The labeled CRPwas purified on a Sephadex G-25 column equilibrated withbuffer A. Fractions displaying a high absorbance at both280 and 340 nm were combined and dialyzed extensivelyagainst buffer A.Mapping of modified residuesMapping of labeled residues was performed as describedpreviously [21] with several modifications. Peptides wereseparated by an HPLC system consisting of (a) a ShimadzuLC-9A pump equipped with FCV-9AL low-pressure pro-portioning valve, (b) a Knauer A0263 manual injectorequipped with a 100-lL loop, (c) a Supelcosil LC-318HPLC (5 lm) cartridge column (250 · 4.6 mm) with20 · 2.1 mm Supelguard LC-318 precolumn, (d) a Merck-Hitachi L-4000A detector, (e) a Shimadzu RF-535 fluores-cence monitor, and (f) a Shimadzu Class-VP 1-2 hardware/software system for data acquisition and analysis. Solvent Awas 0.1% trifluoroacetic acid in water, and solvent B was0.08% trifluoroacetic acid in 80% acetonitrile. A lineargradient of 10–70% solvent B over 40 min was applied at aflow rate of 1 mLÆmin)1, with spectrophotometric detectionat 215 nm and fluorescence detection at an excitationwavelength of 336 nm and an emission wavelength of490 nm.Steady-state fluorescence measurementsSteady-state fluorescence was measured with an HitachiF-4500 spectrofluorimeter. All studies were carried out atroom temperature and excitation at 295 nm. The experi-ments were conducted in buffer A or buffer B. The proteinsolution had an initial absorbance at the excitation wave-length lower than 0.1.The effect of cAMP on tryptophan fluorescence wasmonitored by a fluorescence titration of CRPW13F andCRPW85A. Tryptophan emission was scanned from 310 to480 nm. When energy transfer was measured, the emissionspectra were recorded in the range 310–570 nm. Thefluorescence quantum yield of the donor in the absence ofthe acceptor (QD) was calculated from the equation:QD¼ QRFSDARFSRFADð1Þwhere SDand SRFare the respective areas under theemission spectra of the donor and a reference compound,and ARFand ADare the respective absorbances of thereference compound and donor at the excitation wave-length. QRFis the quantum yield of the reference compoundL-tryptophan and was taken to be 0.14 in water at 25 °C[22].All spectra were corrected for sample dilution and theinner filter effect, introduced by cAMP and DNA atthe excitation wavelength, according to the followingformula [23]Fcor¼ F Â 10PþDAðÞ=2ð2Þwhere F and Fcorare fluorescence intensity before and afterthe correction, and P and DA denote the initial sampleabsorbance at the excitation wavelength and the change inabsorbance introduced by the ligand, respectively.Time-resolved fluorescence measurementsFluorescence decays were measured using a homemadetime-correlated single-photon counting system based onOrtec electronics (Oak Ridge, USA). It consisted of (a) aPhilips 2020Q photomultiplier with a 1.5-ns response time,(b) a 1-GHz preamplifier, (c) a quad constant fractiondiscriminator model 935, and (d) a time-to-amplitudeconverter (TAC) model 457. A nanosecond flash lamp nF900 from Edinburgh Instruments was used as a light source(e). In the case of anisotropy measurements, (f) Glan–Thompson prism polarizers were also used.All measurements were performed at 20 ± 0.2 °C.Before measurements, all samples were filtered through amicroporous filter (0.45 lm; Millipore) to remove insolubleimpurities.FRET measurements. Energy transfer was observedbetween the tryptophan residues and the 1,5-I-AEDANSmoiety covalently attached to Cys178. The tryptophanswere excited at 297 nm. Fluorescence decays were observedat wavelengths between 320 and 400 nm using two cut-offfilters. Measurements were performed in buffer A. Fluor-escence decays were recorded at a resolution of 23 ps perchannel, resulting in a total time window of 100 ns.Intensity decay data were analyzed using the followingmultiexponential decay law:It¼Xiaiexp Àt=siðÞ ð3Þwhere aiand siare the pre-exponential factor and decaytime of component i, respectively. The fractional fluores-cence intensity of each component is defined as ƒi¼ aisi/Saisi. The data were analyzed with the software fromEdinburgh Instruments. Best-fit parameters were obtainedby minimization of the reduced v2value.The average efficiency of energy transfer ÆEæ was calcu-lated from the average donor lifetime in the presence ÆsDAæand absence of acceptor ÆsDæ<E> ¼ 1 À<sDA><sD>ð4ÞÓ FEBS 2003 Conformational changes induced by cAMP and DNA binding to CRP (Eur. J. Biochem. 270) 1415The average lifetime was obtained from the equation:<s> ¼Piais2iPiaisið5ÞAs ÆsDAæ and ÆsDæ are obtained without the need to know theabsolute protein concentrations, uncertainties associatedwith protein concentration determination are eliminated inthe time-resolved fluorescence measurements.The average distance between the donor–acceptor pairÆRæ was calculated from the equation:<R> ¼ R0EÀ1À 1ÀÁ1=6hi½˚AAð6Þwhere R0is the Fo¨rster critical distance (the distance atwhich 50% energy transfer occurs). R0is given by:R0¼ 9:78 Â 103j2nÀ4QDJ kðÞÂÃ1=6½˚AAð7Þwhere n is the refractive index of the medium, QDis thequantum yield of the donor, J(k) is a spectral overlapintegral of the donor fluorescence and acceptor absorption,and j2is the orientation factor and accounts for relativeorientation of the donor emission and acceptor absorptiontransition dipole. Generally, j2isassumedtobeequalto2/3, which is the value for donors and acceptors thatrandomized by rotational diffusion before energy transfer.Fluorescence anisotropy decay measurements. The W13Fand W85A CRP mutants were used to measure therotational correlation time of the protein. The excitationwavelength for tryptophan residues was 297 nm. Fluores-cence anisotropy decays were observed using a cut-offfilter > 320 nm. Experiments were performed at severalconcentrations of the proteins (1.0–8.5 lM) for each species.The sample was excited with vertically polarized light.Fluorescence anisotropy decays with vertical and horizontalemission polarization were alternatively recorded. Allmeasurements were repeated at least twice for each sample.Fluorescence anisotropy decays were recorded at a resolu-tion of 46 ps per channel, resulting in a total time windowof 200 ns.Anisotropy decay data were analyzed according to theimpulse reconvolution model decay law:RtðÞ¼R1þXni¼1Aiexp Àt=hiðÞ ð8Þwhere Aiare the amplitudes of the components withrotational correlation time hI,andRlis limiting anisotropy.The time-zero anisotropy r(0) was obtained from theequation:r 0ðÞ¼R1þXni¼1Aið9ÞIn each case, the best-fit parameters were obtained byminimization of the reduced v2test value. The v2andresiduals distribution were utilized to judge the goodness ofthe fit. The software used for analysis was from EdinburghInstruments.ResultsFluorescence labeling of CRPEach subunit of CRP possesses three cysteine residues, twoin the N-terminal domain (Cys19 and Cys92) and one in theC-terminal domain (Cys178). Only Cys178 can be chemi-cally modified under native conditions; Cys19 and Cys92seem to be buried [24,25]. To confirm the selectivity of thelabeling, CRP mutants modified with 1,5-IAEDANS weredenaturated and completely digested with trypsin andchymotrypsin. The peptides liberated were examined byHPLC. As expected, only one peptide fragment had beenmodified with thiol-reactive probe. Thus, we conclude thatCRP was uniformly labeled with 1,5-IAEDANS at the SHgroup of Cys178.The stoichiometry of the labeling was determined fromthe absorption spectrum of the labeled CRP. WhenCRP was incubated with the fluorescence reagent, 1,5-I-AEDANS, at pH 8.0, a mean of 2 mol was bound permol protein dimer. The effect of the label on thesecondary structure of CRP was investigated using CDspectroscopy. No differences were observed between themodified and unmodified variants of CRP (data notshown). The insertion of the fluorescent probes alsodid not significantly alter the biological activity of CRP[20].Steady-state fluorescence dataThe effect of cAMP on tryptophan fluorescence wasmonitored. Changes in CRP tryptophan fluorescencewere monitored by titrating the CRP solution with 1–2-lL aliquots of concentrated cAMP solution. Measure-ments were performed in the cAMP concentration range50 lMto 1 mM. The fluorescence emission spectra ofTrp13 and Trp85 in the presence and absence of cAMPare given in Fig. 2A and Fig. 3A, respectively. WhenCRP was titrated with cAMP, the fluorescence intensityof the Trp13 residue decreased with increasing ligandconcentration. However, this decrease could only bydetected in the micromolar range of cAMP concentra-tions. In addition, the emission maximum of Trp13shifted from %342.5 nm to %340 nm. The reduction influorescence intensity and the blue shift indicate aconformational transition of CRPW85A on binding ofcAMP. The effect of different concentrations of cAMPon the fluorescence intensity of Trp13 is shown inFig. 2B. When 200 lMcAMP was added to the solutionof CRPW85A, a % 13% decrease in fluorescence intensitywas observed.In contrast with the observation made with CRPW85A,the addition of cAMP to CRPW13F caused an increase inthe fluorescence intensity of Trp85 with no change in theemission maximum. The maximum wavelength of emissionfor CRPW13F was % 339 nm. The effect of differentconcentrations of cAMP on the fluorescence intensity ofTrp85 is shown in Fig. 3B. In the case of Trp85, the additionof 200 lMcAMP caused only a very small change in thefluorescence intensity, increasing it by % 3.4%. A pro-nounced increase in the Trp85 fluorescence intensity was1416 A. Polit et al.(Eur. J. Biochem. 270) Ó FEBS 2003detected only at high concentrations of cAMP (> 2 mM)(data not shown).Typical fluorescence spectra of CRPW13F, unmodifiedand modified with 1,5-I-AEDANS, are shown in Fig. 4.When excited at 295 nm, tryptophan residues in theunlabeled protein had a fluorescence emission maximumnear 339 nm. In the presence of 1,5-I-AEDANS, trypto-phan fluorescence intensity was significantly reduced com-pared with an approximately equal concentration of anunmodified protein. The maximum wavelength of trypto-phan emission in the labeled mutant W13F was shifted to% 327 nm. The addition of cAMP and DNA increasedenergy transfer from Trp85 residue to the AEDANSmoiety.The quantum yields of tryptophan fluorescence at25 °C in buffer A were determined to be 0.09 for mutantCRPW13F alone and 0.094 for mutant CRPW13F in thepresence of 200 lMcAMP. The quantum yield of thedonor increased upon protein–DNA complex formation.The change in the observed value of the quantum yieldwas % 20%. A similar result was obtained for CRP–(cAMP)4.Time-resolved fluorescence dataFRET measurements. Lifetime measurements on themutant CRPW13F labeled with 1,5-I-AEDANS indicateda decreased lifetime of the tryptophan fluorescence, asexpected when energy transfer occurs. Figure 5 shows thetime-dependent donor decays for the proteins bearingdonor alone and those with donor and acceptor. In thecase of mutant CRPW85A, no energy transfer wasobserved. Lifetime measurements were repeated severaltimes for each species. The fluorescence decays wereFig. 3. Fluorescence emission spectra ofCRPW13F in the absence (—) and presence ofcAMP at 50 lM(ÆÆÆÆ) and 1 mMcAMP (- - -).Excitation was at 295 nm. All spectra wererecorded in buffer B, pH 7.8. The inset in theplot shows fluorescence intensity change inTrp85 as a function of cAMP concentration.F and F0are the fluorescence intensities of theprotein in the presence and absence of theligand, respectively. The range of cAMP con-centrations used was from 50 lMto 1 mM.The line was drawn only to indicate the trendof the data.Fig. 2. Fluorescence emission spectra ofCRPW85A in the absence (—) and presence ofcAMP at 50 lM(ÆÆÆÆ) and 1 mMcAMP (- - -).Excitation was at 295 nm. All spectra wererecorded in buffer B, pH 7.8. The inset showsfluorescence intensity change in Trp13 as afunction of cAMP concentration. F and F0arethe fluorescence intensities of the protein in thepresence and absence of the ligand, respect-ively. The range of cAMP concentrations usedwas from 50 lMto 1 mM. The line was drawnonly to indicate the trend of the data.Ó FEBS 2003 Conformational changes induced by cAMP and DNA binding to CRP (Eur. J. Biochem. 270) 1417analyzed as a multiexponential decay, and for each case thedouble exponential decay was characterized by lower valuesof reduced v2.Insomecasesthetripleexponentialdecayswere recorded; however, the pre-exponential factor a3forthese fits was close to zero, and therefore this component ofthe decay is not included in the calculated average values offluorescence lifetimes ÆsDæ and ÆsDAæ. The values for averagelifetimes of Trp85 in the absence and presence of acceptorand efficiency of energy transfer are presented in Table 1.The transfer efficiency varied between apoprotein and afterbinding cAMP at micromolar or millimolar concentrations.In the absence of a specific ligand, CRPW13F showed anefficiency of transfer of 24.2 ± 8.1%. The addition ofcAMP had a significant effect on energy transfer. The valuefor CRPW13F with two cAMP molecules bound to anti-cAMP-binding sites was considerably higher(72.3 ± 2.5%) than for apoprotein. The result determinedfor the mutant W13F in the presence of 2 mMcAMP wassimilar to the above value, averaging 74.3 ± 2.4%. Energytransfer in CRPW13F bound to the specific fragments ofDNA was also measured and displayed similar values ofefficiency for each complex examined (% 62%).The energy-transfer efficiency values were used to calcu-late the average distance between the donor and theacceptor. To approximate the distance between theTrp85–Cys178 pair, we assumed that the Fo¨rster distancefor the tryptophan–IAEDANS pair was 22 A˚[26,27]. Theestimated distances are shown in Table 1. There was asignificant difference between the calculated distance inapo-CRPW13F and CRPW13F complexed with cAMPand DNA.Time-domain anisotropy data. The fluorescence aniso-tropy decays of Trp13 and Trp85 were measured todetermine the changes in the rotational diffusion of CRPafter cAMP binding. The anisotropy decay of Trp13 in theCRP complexed with two cAMP molecules is shown inFig. 6A. Analysis of the anisotropy decays was carried outaccording to Eqn (8) with an increasing number ofexponents, until the fit no longer improved. In all cases,the anisotropy decays of Trp13 and Trp85 could bedescribed by one exponent with single rotational correlationtime. Addition of the second exponent did not significantlyalter the goodness of fit. The v2value obtained with thesingle-exponential and double-exponential analysis indi-cates that the two-component analysis is not significantlybetter than the one-component analysis. Also the distribu-tion of the residuals did not improve on the addition of thesecond component (Fig. 6B,C). However, the data obtainedfor the single exponential analysis suggest the presence of anadditional segmental mobility. This is evident from appar-ent time-zero anisotropy r(0), which is lower than thefundamental anisotropy r0of tryptophan at this excitationwavelength [28]. For both tryptophan residues, the initialanisotropy was in the range 0.22–0.23 (Table 2). It indicatesthat anisotropy decay contains a fast component whichcannot be resolved with our device.The anisotropy decay parameters for various CRPspecies are reported in Table 2. The mean ± SD valueof rotational correlation times determined from the studyof fluorescence anisotropy decays of Trp85 in the absenceof specific ligand was 20.5 ± 2.4 ns. The value for Trp13was considerably lower, averaging 15.3 ± 1.8 ns. Theaddition of 100 lMcAMP probably did not affect therotational correlation time of CRPW13F. The uncer-tainty of this value was too large to ascertain anychanges in the CRP dynamic on cAMP binding. Trp13exhibited different behavior. The rotational correlationtime determined from the study of fluorescence aniso-tropy decays of Trp13 in the presence of cAMPdecreased to 10.45 ± 3.0 ns.DiscussioncAMP binding to CRP has been studied using a variety ofmethods, which have shown that the ligand bindingmediates changes in the protein conformation. It is believedthat these changes allow the protein molecule to switch fromthe low-affinity and nonspecific DNA-binding state to thestate characterized by high affinity and sequence specificityFig. 4. Fluorescence emission spectra ofunmodified and modified CRPW13F. Theexcitation wavelength was 295 nm andemission was scanned from 310 to 570 nm.Measurements were performed at 25 °CinbufferA,pH8.0.(—ÆÆ—) UnmodifiedCRPW13F; (—) modified CRPW13F; (- - -)modified CRPW13F in the presence of 200 lMcAMP; (ÆÆÆÆ) modified CRPW13F bound toDNA in the presence of cAMP.1418 A. Polit et al.(Eur. J. Biochem. 270) Ó FEBS 2003for the DNA promoter [2]. As the X-ray crystal structure ofapo-CRP has not yet been resolved, it is believed that thebinding of cAMP, which leads to a switch to active proteinconformation, involves subunit realignment and hingereorientation between the protein domains [2,29]. For along time, it has been a paradigm that CRP undergoes acAMP concentration-dependent transition between threeconformations: apo-CRP, CRP–(cAMP)1and CRP–(cAMP)2, and each conformer possesses a unique structureand activity [2]. The reinterpretation of this paradigm wasproposed by Passner & Steitz [6] on the basis of the crystalstructure of the CRP–cAMP complex, and it has recentlybeen supported by NMR [7] and isothermal titrationcalorimetry [8] studies in solution. NMR experiments haveshown that CRP possesses two anti-cAMP-binding sites ineach monomer, and the next two syn-cAMP sites areformed by an allosteric conformational change in theprotein on biding of two anti-AMP at the N-terminalFig. 5. Trp85 fluorescence intensity decays for mutant CRPW13F without and with 1,5-I-AEDANS covalently attached to Cys178. The dark greydotted curve shows the intensity decay of the donor alone (D), and the grey dotted curve shows the intensity decay of the donor in the presence ofthe acceptor (DA). The black solid lines and weighted residuals (lower panels) are for the best triple exponential fits. Experiments were performed inbuffer A at 20 °C.Ó FEBS 2003 Conformational changes induced by cAMP and DNA binding to CRP (Eur. J. Biochem. 270) 1419domain [7]. The isothermal titration calorimetry measure-ments demonstrated that, at low cAMP concentration,there are two identical interactive high-affinity sites forcAMP and at least one low-affinity cAMP-binding site athigh concentration of the ligand [8].The idea of the four cAMP-binding sites in CRP, for itsanti conformation (at low concentration of the ligand) andthe next two binding it in syn conformation (at high cAMPconcentration) has been used to describe the results of fastkinetic studies [15] and investigations with dynamic lightscattering and time-resolved fluorescence anisotropy meas-urements [30]. In these studies, we have shown that thebinding of cAMP in anti conformation in the N-terminaldomain of CRP leads to the conformational changes in thehelix–turn–helix motif of the C-terminal domain, respon-sible for the interaction with DNA, as well as to the changesin the global hydrodynamic structure of CRP. The satura-tion of the low-affinity sites with cAMP in syn conformationof cAMP results in the changes in the microenvironment ofthe Trp85 residue, localized in the N-terminal domain of theprotein, without further substantial changes in the globalhydrodynamic structure [30].The results presented in this report provide furtherevidence for conformational changes induced by cAMPbinding to the anti-cAMP-binding sites of CRP, which inturn trigger specific pathways of signal transmission fromthe cyclic nucleotide-binding domain to the DNA-bindingdomain of the protein. We have used single tryptophan-containing mutants of CRP. The mutations were localizedin the N-terminal domain at position 85 or 13 in order tofollow conformational changes in their microenvironmenton binding of cAMP, both in anti- and syn- conformation.We have also used AEDANS for fluorescent labeling ofCys178, located at the turn of the helix–turn–helix motif, inorder to detect FRET between Trp85 and the label. Wehave shown that binding of cAMP at a concentration of200 lMto anti-cAMP-binding sites results in % 13%decrease in the Trp13 fluorescence intensity along with theblue shift in its maximum of emission by 2.5 nm. Probablecandidates for quenching residues in CRP are Thr10,Asn109 and His17, which are located within a distance up to5A˚, as has been determined from the X-ray crystalstructure of the CRP–cAMP complex (PDB code 1G6N)[29]. The observed changes in the microenvironment ofTrp13 on filling of the high-affinity sites are also supportedby the time-resolved anisotropy measurements. Binding ofthe ligand to anti-cAMP-binding sites results in a decrease inrotational correlation time by %5 ns, from the value of15.3 ns, detected for apo-CRP, to the value of 10.4 ns forCRP–(cAMP)2complex. The decrease in rotational timeindicates an increase in the mobility of helix A of theprotein. As Trp13 is located in the vicinity of the activationregion AR2 of the protein [1], which is responsible for theactivation of the second class of E. coli promoters such asgal P1, one can speculate that this conformational changemay play an important role in a signal transmission in theprotein molecule, which in turn may allow CRP to adopt aconformation appropriate for the interaction with theaNTD domain of RNA polymerase in the transcriptioncomplex. On the other hand, Trp13 of CRP directlyinteracts with another gene regulatory protein, CytR [31],and the observed conformational changes in Trp13 micro-environments on cAMP binding to anti-cAMP sites mayplay a significant role in the CRP–CytR–DNA complex.In contrast with Trp13 of CRP, binding of cAMP to anti-cAMP-binding sites does not lead to a significant change influorescence intensity of Trp85, and only a % 3.4% increasein the intensity has been observed at 200 lMcAMP.However, cAMP binding to the syn-cAMP-binding sites atconcentration of the ligand of 1 mMcauses a % 6% increasein its fluorescence intensity, which indicates that this residueis sensitive to cAMP binding to low-affinity sites. Therotational correlation time of Trp85 in apo-CRP of 20.5 nsindicates that this residue is immobilized within theN-terminal domain of the protein and exhibits motioncharacteristic of the whole protein (within experimentalerror), while the respective value for the AEDANS-labeledapo-CRP has been estimated at 23.3 ns [30]. Binding ofcAMP to anti-cAMP-binding sites increases the rotationalcorrelation time to 22 ns for the CRP–(cAMP)2complex,which is much lower than the correlation time of 30 nsdetermined for this complex using CRP labeled at Cys178with the AEDANS fluorescent probe [30]. These discre-pancies can probably be explained by the fact that theaverage fluorescence lifetime of Trp85, % 5.6 ns in the caseof the CRP–(cAMP)2complex, is too short to allowobservations of the longer rotational correlation times.The allosteric activation of CRP involves conformationalchanges in the N-terminal domain of the protein and leadsto changes in the CRP molecule, enabling it to recognize thespecific DNA sequence [1,2]. As the crystal structure of apo-CRP has not yet been established, it was suggested thatcAMP binding may cause reorientation of the coiled-coil Chelices, consequently altering the relative position of theprotein dimer subunits [29]. These authors also suggestedthat, in the absence of cAMP in apo-CRP, some b strandsof the N-terminal domain of the protein may collapse intothe cAMP-binding pocket, causing reorientation of thesmaller domain in relation to the larger one, and bringingthese domains closer together. This suggestion has beenTable 1. Summary of energy transfer measurements. In the CRPW13F–(cAMP)2complex, the concentration of cAMP was 200 lM,whereasinthecase of CRPW13F–(cAMP)4the concentration of cAMP was 2 mM. The molar ratio CRP to DNA in the protein–DNA complex was 1 : 1.Species ÆsDæ (ns) ÆsDAæ (ns) ÆEæ (%) ÆRæ (A˚)CRPW13F 5.83 ± 0.50 4.42 ± 0.28 24.2 ± 8.1 26.6 ± 3.9CRPW13F–(cAMP)25.59 ± 0.31 1.55 ± 0.11 72.3 ± 2.5 18.7 ± 0.8CRPW13F–(cAMP)45.99 ± 0.42 1.54 ± 0.10 74.3 ± 2.4 18.4 ± 0.8CRPW13F–ICAP 5.55 ± 0.25 2.10 ± 0.06 62.2 ± 2.0 20.2 ± 0.6CRPW13F–lac 5.85 ± 0.16 2.23 ± 0.06 61.9 ± 1.5 20.3 ± 0.5CRPW13F–gal 5.85 ± 0.12 2.17 ± 0.25 62.9 ± 4.3 20.1 ± 1.21420 A. Polit et al.(Eur. J. Biochem. 270) Ó FEBS 2003supported recently by NMR studies [7]; these authors arguethat binding in solution of two cAMP molecules to high-affinity anti-cAMP-binding sites at the N-terminal domaincauses the C-terminal domain to shift further to theN-terminal domain of CRP. To confirm this suggestion,we used FRET to detect the distance between the C-terminaland N-terminal domains of CRP on binding of cAMP in antias well as syn conformation to the protein. For this purpose,we used time-resolved fluorescence lifetime measurementsTable 2. Parameters of Trp13 and Trp85 anisotropy decays in thepresence and absence of cAMP.Species h (ns) r(0) v2CRPW13F 20.5 ± 2.4 0.23 ± 0.02 1.222CRPW85A 15.3 ± 1.8 0.21 ± 0.02 1.055CRPW13F–(cAMP)222.1 ± 6.9 0.23 ± 0.03 1.036CRPW85A–(cAMP)210.45 ± 3.0 0.23 ± 0.03 1.146Fig. 6. Time-domain fluorescence anisotropy decay of Trp13 in the presence of 100 lMcAMP. The solid line corresponds to the best singleexponential fit of the data (dotted curve) according to Eqn (8). The grey cross-haired curve represents the lamp profile. The plots of the residuals forthe best single exponential fit (B) and the double exponential fit (C) are also shown. Measurements were performed at 20 °C in buffer B, pH 8.0,with a CRPW85A concentration of 1.1 lM. Excitation was at 297 nm.Ó FEBS 2003 Conformational changes induced by cAMP and DNA binding to CRP (Eur. J. Biochem. 270) 1421using single tryptophan-containing mutants of CRP. Fluo-rescence energy transfer could be detected between Trp85,localized close to the cAMP-binding pocket of theN-terminal domain, and Cys178, fluorescently labeled byAEDANS, localized in the helix–turn–helix motif of theC-terminal domain of the protein. The lifetimes obtained forthe fluorescence donor, Trp85, indicate that binding ofcAMP to anti-cAMP-binding sites leads to a dramaticincrease in FRET efficiency. This observation clearly shows adecrease in the average distance between the two domains ofCRP on cAMP binding. If one assumes the Fo¨rster distance,R0, for the pair donor–acceptor such as tryptophan–AEDANS to be 22 A˚[26,27], the distance between Trp85and Cys178-AEDANS in the apo-CRP can be calculated tobe 26.6 A˚. This distance decreases by about 8 A˚to 18.7 A˚onbinding of cAMP to the anti-cAMP-binding sites of theprotein. The distance between the sulfur atom of Cys178 andthe C9–C10 bond of the indole ring of Trp85, derived fromthe crystal structure of CRP–(cAMP)2(PDB code 1G6N) is18.9 A˚and 21.9 A˚for the subunit present in the ÔclosedÕ andÔopenÕ conformation, respectively [29]. The structural asym-metry of the CRP–(cAMP)2complex resulting from con-formational differences between subunits has beenquestioned [32], and from molecular dynamics simulation,it has been predicted that, in solution, both subunits of CRPadopt a ÔclosedÕ conformation. If this is so, the distance (equalto 18.7 A˚), determined in this work by FRET, is in goodagreement with the value of 18.9 A˚predicted for the ÔclosedÕconformation. This supports experimentally the dynamicsimulation studies [32] and indicates that, in solution, both ofthe protein subunits exist in ÔclosedÕ conformation in theCRP–(cAMP)2complex.Because a variety of spectroscopic effects, at least intheory, could influence energy transfer efficiency, one canargue that the good agreement determined for the distancebetween Cys178 and Trp85 residues in CRP–(cAMP)2mayalso result from the assumed value of Fo¨rster distance R0.However, as binding of cAMP at a concentration of 200 lMto CRP leads only to a % 4.3% increase in the fluorescencequantum yields from the value of 0.09 for apo-CRP to thevalue of 0.094 for the CRP–(cAMP)2complex, and nosubstantial changes in the shape of the emission spectra ofthe donor have been observed, this justifies the lack of thealteration of the Fo¨rster distance between apo and holoforms of the protein. As the AEDANS label attached to theCys178 enjoys local freedom of movement, in both apo-CRP and the CRP–(cAMP)2complex [30], the distanceobtained from the crystal structure between the sulfur atomof Cys178 and the indole ring of Trp85 seems to be realistic.We have also tried to measure fluorescence energy transferbetween Trp13 and Cys178-AEDANS; however, we havenot detected any energy transfer in either the apo- or holo- form. This could be because of the distance between the tworesidues, which is about 45 A˚, as can be calculated from thecrystal structure of CRP–(cAMP)2[29].Binding of cAMP in syn conformation to the low-affinity binding sites in the CRP–(cAMP)4complex leadsto only a small increase in the efficiency of energytransfer,which,withanassumedR0value of 22 A˚,corresponds to the small decrease in average distancebetween the N-terminal and C-terminal domains of CRP,estimated at 18.4 A˚. However, the fluorescence quantumyield of the Trp85 donor increases by % 20% at aconcentration of cAMP of 2 mMfrom the valuecharacteristic of apo-CRP, which in turn may beresponsible for this very small change. We have alsomeasured the distance between the two domains of CRPin the complexes with DNA containing various sequenc-es, such as lac and gal promoters and with the symmetricsequence ICAP. For each CRP–DNA complex, theincrease in the distance between the two CRP domainshas been observed with the average distance of 20.2 A˚.This value is in good agreement with the value of20.7 A˚, calculated from the crystal structure of the CRP–DNA complex [33].The present results show that the binding of anti-cAMPin the CRP–(cAMP)2complex results in the movement ofthe C-terminal domain of CRP by % 8A˚towards theN-terminal domain, which in consequence leads torearrangement of DNA-binding domains and cAMP-binding domains of the protein. This finding clarifies thesuggestion derived from the NMR measurements [7] thatthe C-terminus is closer to the N-terminal domain in apo-CRP than in cAMP-bound CRP. Binding of cAMP toanti-cAMP-binding sites leads to an increase in thestructural dynamic motion around Trp13, which is closeto the activation region AR2, responsible for the interac-tion of CRP with the a subunit of RNA polymerase. Thechanges in the CRP dynamics on cAMP binding haverecently been observed by the hydrogen exchange method[34]. In that paper, it was shown that binding of theligand to the protein causes the C-terminal domain ofCRP to become more flexible, in contrast with the N-terminal domain which is shifted to a less dynamicconformation. Our results extend this observation andsuggest that the binding of cAMP to anti-cAMP-bindingsites of CRP leads to the increase in the structuraldynamic motion of at least Trp13, which is located in theN-terminal domain of the protein.AcknowledgementsWe are grateful to Dr S. Garges for supplying us with the plasmid forproduction of CRP. This work was supported by grant no.6 P04A 031 16 from the State Committee for Scientific Research.References1. Busby, S. & Ebright, R. (1999) Transcription activation by Cata-bolite Activator Protein (CAP). J. Mol. Biol. 293, 199–213.2. Harman, J.G. (2001) Allosteric regulation of the cAMP receptorprotein. Biochim. Biophys. Acta 2, 1–17.3. de Crombrugghe, B., Busby, S. & Buc, H. (1984) Cyclic AMPreceptor protein: role in transcription activation. Science 224,831–838.4. Weber, I.T. & Steitz, T.A. (1987) Structure of a complex of cata-bolite gene activator protein and cyclic AMP refined at 2.5 A˚resolution. J. Mol. Biol. 198, 311–326.5. Parkinson, G., Wilson, C., Gunasekera, A., Ebright, Y.W.,Ebright, R.E. & Berman, H. (1996) Structure of the CAP–DNAcomplex at 2.5 A˚resolution: a complete picture of the protein–DNA interface. J. Mol. Biol. 260, 395–408.6. Passner, J.M. & Steitz, T.A. (1997) The structure of a CAP–DNAcomplex having two cAMP molecules bound to each monomer.Proc. Natl. Acad. Sci. USA 94, 2843–2847.1422 A. Polit et al.(Eur. J. Biochem. 270) Ó FEBS 2003[...]... 3-fluorotyrosine-containing cyclic AMP receptor protein from Escherichia coli J Biochem 266, 545–552 14 Wasylewski, M., Małecki, J & Wasylewski, Z (1995) Fluorescence study of Escherichia coli cyclic AMP receptor protein J Protein Chem 14, 299–308 15 Małecki, J., Polit, A & Wasylewski, Z (2000) Kinetic studies of cAMP -induced allosteric changes in cyclic AMP receptor protein from Escherichia coli J Biol Chem... 2003 Conformational changes induced by cAMP and DNA binding to CRP (Eur J Biochem 270) 1423 7 Won, Y.-S., Lee, T.-W., Park, S.-H & Lee, B.-J (2002) Stoichiometry and effect of the cyclic nucleotide binding to cyclic AMP receptor protein J Biol Chem 277, 11450–11455 8 Lin, S.-H & Lee, J.C (2002) Communications between the highaffinity cyclic nucleotide binding sites in E coli cyclic AMP receptor protein: ... roles of the two cyclic AMP- dependent forms of cyclic AMP receptor protein from Escherichia coli FEBS Lett 453, 215–218 12 McKay, D.B & Steitz, T.A (1981) Structure of catabolite gene ˚ activator protein at 2.9 A resolution suggests binding to lefthanded B -DNA Nature (London) 290, 744–749 13 Sixl, F., King, R.W., Bracken, M & Feeney, J (1990) 19F-n.m.r studies of ligand binding to 5-fluorotryptophan- and. .. protein: effect of single site mutations Biochemistry 41, 11857–11867 9 Heyduk, T & Lee, J.S (1989) Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities Biochemistry 28, 6914–6924 10 Taniguchi, T., O’Neill, M & de Crombrugghe, B (1979) Interaction site of Escherichia coli cyclic AMP receptor protein on DNA of galactose operon promoters... freedom of tryptophan residues in proteins and peptides Biochemistry 22, 1741–1752 29 Passner, J.M., Schultz, S.C & Steitz, T.A (2000) Modelling the cAMP -induced allosteric transition using the crystal ˚ structure of CAP-cAMP at 2.1 A resolution J Mol Biol 304, 847–859 30 Błaszczyk, U., Polit, A., Guz, A & Wasylewski, Z (2002) Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA. .. study of the cooperative binding of adenosine cyclic 3¢,5¢-monophosphate and guanosine cyclic 3¢,5¢-monophosphate to the adenosine cyclic 3¢,5¢-monophosphate receptor protein from Escherichia coli Biochemistry 19, 5124–5130 18 Hudson, E.N & Weber, G (1973) Synthesis and characterization of two fluorescent sulfhydryl reagents Biochemistry 12, 4154– 4161 19 Gill, S.C & von Hippel, P.H (1989) Calculation of. .. Biochemistry 30, 2707–2712 22 Wu, P & Brand, L (1994) Resonance energy transfer: methods and applications Anal Biochem 218, 1–13 23 Lakowicz, J.R (1999) Principles of Fluorescence Spectroscopy Kluwer Academic Publisher, Dordrecht, the Netherlands 24 Eilen, E & Krakow, J.S (1977) Cyclic AMP- mediated intersubunit disulfide crosslinking of the cyclic AMP receptor protein of Escherichia coli J Mol Biol 114, 47–60 25... Hippel, P.H (1989) Calculation of protein coefficients from amino acids sequence data Anal Biochem 182, 319– 326 20 Wu, F.Y.-H., Nath, K & Wu, C.-W (1974) Conformational transitions of cyclic adenosine monophosphate receptor protein of Escherichia coli A fluorescence probe study Biochemistry 13, 2567–2572 21 Gardner, J.A & Matthews, K.S (1991) Energy transfer in lactose repressor protein modified with N-[[(iodoactyl)... Simulations of CRP: (cAMP)2 in noncrystalline environments show a subunits transition from the open to closed conformation Protein Sci 5, 62–71 33 Schultz, S.C., Shields, G.C & Steitz, T.A (1991) Crystal structure of a CAP DNA complex: the DNA is bent by 90 degrees Science 253, 1001–1007 34 Dong, A., Małecki, J.M., Lee, L., Carpenter, J.F & Lee, J.C (2002) Ligand -induced conformational and structural... Escherichia coli with cAMP and DNA studied by dynamic light scattering and timeresolved fluorescence anisotropy methods J Protein Chem 20, 601–610 31 Søgaard-Andersen, L., Mironov, A.S., Pedersen, H., Sukhodelets, V.V & Valentin-Hansen, P (1991) Single amino acid substitutions in the cAMP receptor protein specifically abolish regulation by the CytR repressor in Escherichia coli Proc Natl Acad Sci USA 88, 4921–4925 . Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia. & Feeney, J. (1990)19F-n.m.r. studies of ligand binding to 5-fluorotryptophan- and 3-fluoroty-rosine-containing cyclic AMP receptor protein from Escherichia coli.
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