Eur J Biochem 270, 1413–1423 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03497.x 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 Agnieszka Polit, Urszula Błaszczyk and Zygmunt Wasylewski Department of Physical Biochemistry, Faculty of Biotechnology, Jagiellonian University, Krako´w, Poland cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached to Cys178 Binding of cAMP in the CRP–(cAMP)2 complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP– (cAMP)4 complex alters the surroundings of Trp85 Timeresolved anisotropy measurements indicated that cAMP binding in the CRP–(cAMP)2 complex led to a substantial increase in the rotational mobility of the Trp13 residue Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP–(cAMP)2 complex caused a substantial increase in FRET efficiency This indicates a decrease in the distance between the two domains of the protein from ˚ ˚ 26.6 A in apo-CRP to 18.7 A in the CRP–(cAMP)2 complex The binding of cAMP in the CRP–(cAMP)4 complex resulted in only a very small increase in FRET efficiency The average distance between the two domains in CRP–DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance ˚ between Cys178 and Trp85 to % 20 A The spectral changes observed provide new structural information about the cAMP-induced allosteric activation of the protein cAMP receptor protein (CRP), which is allosterically activated by cAMP, regulates transcription of over 100 genes in Escherichia coli [1,2] Upon binding the cyclic nucleotide, CRP undergoes an allosteric conformational change that allows it to bind specific DNA sequences with increased affinity [3] CRP is a dimeric protein, composed of two identical 209-amino-acid subunits Each subunit of CRP has a molecular mass of 23.6 kDa, as deduced from the amino-acid sequence Individual subunits fold into two domains [4] The larger N-terminal domain (residues 1–133) is responsible for dimerization of CRP and for interaction with the allosteric effector, cAMP The smaller C-terminal domain (residues 139–209) is responsible for interaction with DNA through a helix–turn–helix motif CRP recognizes a 22-bp, symmetric DNA site [5] Amino-acid residues 134–138 form a flexible hinge which covalently couples two domains Recent studies of the crystal structure of the CRP– DNA complex showed that each protein subunit binds two cAMP molecules with different affinities [6] Higher-affinity sites, where the nucleotide binds in the anti conformation, are buried within the N-terminal domains, whereas loweraffinity binding sites (where the bound cAMP has a syn conformation) are located at the interface formed by the two C-terminal domains of the CRP subunits, interacting with a helix–turn–helix motif and, indirectly, with the DNA Crystallographic observations have been supported by recent NMR [7] and isothermal titration calorimetry studies [8] Therefore, it has been suggested that CRP exists in three conformational states: free CRP, CRP with two cAMP molecules bound to N-terminal domains [CRP–(cAMP)2], and CRP with four cAMP molecules bound to both N-terminal and C-terminal domains [CRP–(cAMP)4] An earlier hypothesis suggested [9] that the three conformational states of CRP consisted of the following species: free CRP, CRP–(cAMP)1 and CRP–(cAMP)2, which has been reinterpreted by Passner & Steitz [6] It is important to note that the behavior of CRP at different concentrations of cAMP is essentially biphasic, so two different conformers exist at lower and higher concentrations of cAMP In the presence of % 100 lM cAMP, CRP becomes activated and is able to recognize and bind specific DNA sequences and stimulate transcription [10], whereas at millimolar concentrations of cAMP, there is a loss of affinity and sequence specificity for DNA binding and, consequently, loss of transcription stimulation [11] In the crystal phase, the CRP Correspondence to Z Wasylewski, Department of Physical Biochemistry, Faculty of Biotechnology, Jagiellonian University, ´ ul Gronostajowa 7, 30-387 Krakow, Poland Fax: + 48 12 25 26 902, Tel.: + 48 12 25 26 122, E-mail: wasylewski@mol.uj.edu.pl Abbreviations: 1,5-I-AEDANS, N-iodoacetylaminoethyl-1-naphthylamine-5-sulfonate; AEDANS-CRP, CRP covalently labeled with 1,5-I-AEDANS attached to Cys178; apo-CRP, unligated CRP; CRP, cAMP receptor protein; FRET, fluorescence resonance energy transfer (Received 31 October 2002, revised 19 December 2002, accepted February 2003) Keywords: allosteric regulation; cAMP receptor protein; emission anisotropy; Escherichia coli; fluorescence Ó FEBS 2003 1414 A Polit et al (Eur J Biochem 270) cAMP mediates the allosteric activation of CRP remain obscure, because the crystal structure of apo-CRP has not yet been elucidated Therefore, we decided to study the changes in the apo-CRP structure induced by binding of cAMP and DNA by measuring fluorescence resonance energy transfer (FRET) and fluorescence anisotropy decay Two tryptophan residues, Trp13 and Trp85, were used as intrinsic donors for FRET analysis The fluorescence anisotropy decay was used to investigate the dynamics of CRP Experimental procedures Materials Fig Structure of the CRP dimer The locations of tryptophan residues are marked in red, and the location of Cys178 residue is indicated in yellow The figure was generated with WEBLAB VIEWERPRO (version 3.7) using atomic coordinates for the CRP–cAMP complex [29] The coordinates were obtained from the Brookhaven Protein Data Bank (accession code 1G6N) dimer with two molecules of cAMP bound to anti-cAMP binding sites is asymmetric, i.e one monomer is in an ÔopenÕ form, in which the a-helices are swung out away from the N-terminal domain, and the other monomer is in a ÔclosedÕ form, in which the a-helices are swung in close to the N-terminal domain [4] X-ray crystal structure studies revealed that the cAMP-ligated CRP dimer complexed to a 30-bp DNA sequence is exclusively in the ÔclosedÕ form [12] Each subunit of CRP contains two tryptophan residues at positions 13 and 85 (Fig 1) Both residues are located in the N-terminal domain, Trp85 near the cAMP-binding pocket and Trp13 on the surface of the protein Trp13 is much more accessible to solvent than Trp85 19F-NMR studies have revealed that binding of cAMP induces not only changes in the immediate environment of the cAMP-binding site but also far-reaching conformational changes (perturbation of chemical shift of Trp13) [13] Fluorescence studies have shown that Trp13 is responsible for % 80% of the tryptophan fluorescence in CRP with % 20% of the signal originating from Trp85 [14] Acrylamide-mediated and iodide-mediated fluorescence quenching studies indicate that Trp13 is solventexposed and accessible to the quenching agents On the other hand, Trp85 is inaccessible to quenching agents Heyduk & Lee [9] have shown that, at micromolar cAMP concentrations, there is no detectable change in fluorescence intensity of protein tryptophan residues The increase was only detected in the millimolar range of cAMP concentration Biochemical and biophysical studies have demonstrated that binding of cAMP allosterically induces CRP to assume a conformation that binds to DNA and interacts with RNA polymerase However, the details of the mechanism by which Acrylamide, KCl, EDTA, phenylmethanesulfonyl fluoride, Tris and N-iodoacetylaminoethyl-1-naphthylamine-5-sulfonate (1,5-I-AEDANS) were purchased from Sigma cAMP and dithiothreitol were from Fluka The Fractogel EMD – SO3 650 (M) was from Merck, and Q-Sepharose Fast Flow, Sephacryl S-200 HR and Sephadex G-25 were from Amersham Pharmacia Biotech DNA sequence containing ´ the CRP-binding sites was from TIB MOLBIOL (Poznan, Poland) The nutrients for bacterial growth were from Life Technologies All other chemicals were analytical-grade products of POCh-Gliwice (Gliwice, Poland) All measurements were performed in buffers prepared in water purified with the Millipore system The sequences of duplexes used in this study were as follows: lac (26 bp), 5¢-ATTAATGTGAGTTAGCTCACTCATT A-3¢ and 3¢-TAATTACACTCAATCGAGTGAGTAAT-5¢; gal (26 bp), 5¢-AAAAGTGTGACATGGAATAAATT AGT-3¢ and 3¢-TTTTCACACTGTACCTTATTTAATCA-5¢; ICAP (28 bp), 5¢-AATTAATGTGACATATGTCACAT TAATT-3¢ and 3¢-TTAATTACACTGTATACAGTGTAAT TAA-5¢ The recognition half-sites are shown in bold An equimolar amount of complementary strand was added, and the mixture was heated for at 96 °C and slowly cooled to room temperature The double-stranded DNA was stored at )20 °C in experimental buffer Protein purification The tryptophans at positions 13 and 85 of CRP were replaced with phenylalanine and alanine, respectively The mutagenesis was performed using the overlap extension method with Pwo DNA polymerase pHA7 plasmid encoding mutant crp genes was introduced into E coli strain M182Dcrp, kindly provided by Dr S Busby (The University of Birmingham, UK) The bacteria were grown on Luria–Bertani medium at 37 °C overnight in a Biostat B fermentor from B Braun Biotech International (Melsungen, Germany) Proteins were purified at °C, essentially as described previously [15] but with one modification After ion-exchange chromatography on Q-Sepharose, the proteins were additionally purified by gel 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: 14 650 )1 )1 M Ỉcm at 259 nm for cAMP [16] and 6000 M)1Ỉcm)1 at Ĩ FEBS 2003 Conformational changes induced by cAMP and DNA binding to CRP (Eur J Biochem 270) 1415 340 nm for AEDANS [18] Absorption coefficients of CRP mutants were determined using the method described elsewhere [19] as 29 700 M)1Ỉcm)1 and 33 100 M)1Ỉcm)1 at 278 nm for the W13F and W85A dimers, respectively Measurements were performed in 50 mM Tris/HCl buffer, pH 8.0, containing 100 mM KCl and mM EDTA (buffer A), and 50 mM Tris/HCl buffer, pH 7.8, supplemented with 100 mM KCl and mM EDTA (buffer B) Fluorescence labeling of CRP Covalent modification of Trp mutants with 1,5-I-AEDANS was carried out as described elsewhere [20] with several modifications The protein and label were mixed at a molar ratio of : 10 and incubated at room temperature for h, and then at °C overnight in the dark The labeled CRP was purified on a Sephadex G-25 column equilibrated with buffer A Fractions displaying a high absorbance at both 280 and 340 nm were combined and dialyzed extensively against buffer A Mapping of modified residues Mapping of labeled residues was performed as described previously [21] with several modifications Peptides were separated by an HPLC system consisting of (a) a Shimadzu LC-9A pump equipped with FCV-9AL low-pressure proportioning valve, (b) a Knauer A0263 manual injector equipped with a 100-lL loop, (c) a Supelcosil LC-318 HPLC (5 lm) cartridge column (250 · 4.6 mm) with 20 · 2.1 mm Supelguard LC-318 precolumn, (d) a MerckHitachi L-4000A detector, (e) a Shimadzu RF-535 fluorescence monitor, and (f) a Shimadzu Class-VP 1-2 hardware/ software system for data acquisition and analysis Solvent A was 0.1% trifluoroacetic acid in water, and solvent B was 0.08% trifluoroacetic acid in 80% acetonitrile A linear gradient of 10–70% solvent B over 40 was applied at a flow rate of mLỈmin)1, with spectrophotometric detection at 215 nm and fluorescence detection at an excitation wavelength of 336 nm and an emission wavelength of 490 nm Steady-state fluorescence measurements Steady-state fluorescence was measured with an Hitachi F-4500 spectrofluorimeter All studies were carried out at room temperature and excitation at 295 nm The experiments were conducted in buffer A or buffer B The protein solution had an initial absorbance at the excitation wavelength lower than 0.1 The effect of cAMP on tryptophan fluorescence was monitored by a fluorescence titration of CRPW13F and CRPW85A Tryptophan emission was scanned from 310 to 480 nm When energy transfer was measured, the emission spectra were recorded in the range 310–570 nm The fluorescence quantum yield of the donor in the absence of the acceptor (QD) was calculated from the equation: QD ¼ QRF SD ARF SRF AD ð1Þ where SD and SRF are the respective areas under the emission spectra of the donor and a reference compound, and ARF and AD are the respective absorbances of the reference compound and donor at the excitation wavelength QRF is the quantum yield of the reference compound L-tryptophan and was taken to be 0.14 in water at 25 °C [22] All spectra were corrected for sample dilution and the inner filter effect, introduced by cAMP and DNA at the excitation wavelength, according to the following formula [23] Fcor ¼ F 10PỵDAị=2 2ị where F and Fcor are uorescence intensity before and after the correction, and P and DA denote the initial sample absorbance at the excitation wavelength and the change in absorbance introduced by the ligand, respectively Time-resolved fluorescence measurements Fluorescence decays were measured using a homemade time-correlated single-photon counting system based on Ortec electronics (Oak Ridge, USA) It consisted of (a) a Philips 2020Q photomultiplier with a 1.5-ns response time, (b) a 1-GHz preamplifier, (c) a quad constant fraction discriminator model 935, and (d) a time-to-amplitude converter (TAC) model 457 A nanosecond flash lamp nF 900 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 a microporous filter (0.45 lm; Millipore) to remove insoluble impurities FRET measurements Energy transfer was observed between the tryptophan residues and the 1,5-I-AEDANS moiety covalently attached to Cys178 The tryptophans were excited at 297 nm Fluorescence decays were observed at wavelengths between 320 and 400 nm using two cut-off filters Measurements were performed in buffer A Fluorescence decays were recorded at a resolution of 23 ps per channel, resulting in a total time window of 100 ns Intensity decay data were analyzed using the following multiexponential decay law: X It ¼ exp ðÀt=si Þ ð3Þ i where and si are the pre-exponential factor and decay time of component i, respectively The fractional fluorescence intensity of each component is defined as ƒi ¼ aisi/ Saisi The data were analyzed with the software from Edinburgh Instruments Best-fit parameters were obtained by minimization of the reduced v2 value The average efficiency of energy transfer Ỉ was calculated from the average donor lifetime in the presence ỈsD and absence of acceptor ỈsDỉ ¼ À < sDA > < sD > ð4Þ Ó FEBS 2003 1416 A Polit et al (Eur J Biochem 270) The average lifetime was obtained from the equation: P si i ẳ P 5ị a i si i As ỈsD and ỈsDỉ are obtained without the need to know the absolute protein concentrations, uncertainties associated with protein concentration determination are eliminated in the time-resolved fluorescence measurements The average distance between the donor–acceptor pair ỈRỉ was calculated from the equation: hÀ Á1=6 i ˚ < R > ẳ R0 E1 ẵA 6ị where R0 is the Forster critical distance (the distance at ă which 50% energy transfer occurs) R0 is given by:  Ã1=6 ẵA 7ị R0 ẳ 9:78 103 j2 n4 QD JðkÞ where n is the refractive index of the medium, QD is the quantum yield of the donor, J(k) is a spectral overlap integral of the donor fluorescence and acceptor absorption, and j2 is the orientation factor and accounts for relative orientation of the donor emission and acceptor absorption transition dipole Generally, j2 is assumed to be equal to 2/3, which is the value for donors and acceptors that randomized by rotational diffusion before energy transfer Fluorescence anisotropy decay measurements The W13F and W85A CRP mutants were used to measure the rotational correlation time of the protein The excitation wavelength for tryptophan residues was 297 nm Fluorescence anisotropy decays were observed using a cut-off filter > 320 nm Experiments were performed at several concentrations 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 horizontal emission polarization were alternatively recorded All measurements were repeated at least twice for each sample Fluorescence anisotropy decays were recorded at a resolution of 46 ps per channel, resulting in a total time window of 200 ns Anisotropy decay data were analyzed according to the impulse reconvolution model decay law: Rtị ẳ R1 ỵ n X Ai exp t=hi ị 8ị iẳ1 where Ai are the amplitudes of the components with rotational correlation time hI, and Rl is limiting anisotropy The time-zero anisotropy r(0) was obtained from the equation: r0ị ẳ R1 ỵ n X Ai 9ị iẳ1 In each case, the best-fit parameters were obtained by minimization of the reduced v2 test value The v2 and residuals distribution were utilized to judge the goodness of the fit The software used for analysis was from Edinburgh Instruments Results Fluorescence labeling of CRP Each subunit of CRP possesses three cysteine residues, two in the N-terminal domain (Cys19 and Cys92) and one in the C-terminal domain (Cys178) Only Cys178 can be chemically modified under native conditions; Cys19 and Cys92 seem to be buried [24,25] To confirm the selectivity of the labeling, CRP mutants modified with 1,5-IAEDANS were denaturated and completely digested with trypsin and chymotrypsin The peptides liberated were examined by HPLC As expected, only one peptide fragment had been modified with thiol-reactive probe Thus, we conclude that CRP was uniformly labeled with 1,5-IAEDANS at the SH group of Cys178 The stoichiometry of the labeling was determined from the absorption spectrum of the labeled CRP When CRP was incubated with the fluorescence reagent, 1,5I-AEDANS, at pH 8.0, a mean of mol was bound per mol protein dimer The effect of the label on the secondary structure of CRP was investigated using CD spectroscopy No differences were observed between the modified and unmodified variants of CRP (data not shown) The insertion of the fluorescent probes also did not significantly alter the biological activity of CRP [20] Steady-state fluorescence data The effect of cAMP on tryptophan fluorescence was monitored Changes in CRP tryptophan fluorescence were monitored by titrating the CRP solution with 1–2lL aliquots of concentrated cAMP solution Measurements were performed in the cAMP concentration range 50 lM to mM The fluorescence emission spectra of Trp13 and Trp85 in the presence and absence of cAMP are given in Fig 2A and Fig 3A, respectively When CRP was titrated with cAMP, the fluorescence intensity of the Trp13 residue decreased with increasing ligand concentration However, this decrease could only by detected in the micromolar range of cAMP concentrations In addition, the emission maximum of Trp13 shifted from % 342.5 nm to % 340 nm The reduction in fluorescence intensity and the blue shift indicate a conformational transition of CRPW85A on binding of cAMP The effect of different concentrations of cAMP on the fluorescence intensity of Trp13 is shown in Fig 2B When 200 lM cAMP was added to the solution of CRPW85A, a % 13% decrease in fluorescence intensity was observed In contrast with the observation made with CRPW85A, the addition of cAMP to CRPW13F caused an increase in the fluorescence intensity of Trp85 with no change in the emission maximum The maximum wavelength of emission for CRPW13F was % 339 nm The effect of different concentrations of cAMP on the fluorescence intensity of Trp85 is shown in Fig 3B In the case of Trp85, the addition of 200 lM cAMP caused only a very small change in the fluorescence intensity, increasing it by % 3.4% A pronounced increase in the Trp85 fluorescence intensity was Ó FEBS 2003 Conformational changes induced by cAMP and DNA binding to CRP (Eur J Biochem 270) 1417 Fig Fluorescence emission spectra of CRPW85A in the absence (—) and presence of cAMP at 50 lM (ỈỈỈỈ) and mM cAMP (- - -) Excitation was at 295 nm All spectra were recorded in buffer B, pH 7.8 The inset shows fluorescence intensity change in Trp13 as a function of cAMP concentration F and F0 are the fluorescence intensities of the protein in the presence and absence of the ligand, respectively The range of cAMP concentrations used was from 50 lM to mM The line was drawn only to indicate the trend of the data Fig Fluorescence emission spectra of CRPW13F in the absence (—) and presence of cAMP at 50 lM (ỈỈỈỈ) and mM cAMP (- - -) Excitation was at 295 nm All spectra were recorded in buffer B, pH 7.8 The inset in the plot shows fluorescence intensity change in Trp85 as a function of cAMP concentration F and F0 are the fluorescence intensities of the protein in the presence and absence of the ligand, respectively The range of cAMP concentrations used was from 50 lM to mM The line was drawn only to indicate the trend of the data detected only at high concentrations of cAMP (> mM) (data not shown) Typical fluorescence spectra of CRPW13F, unmodified and modified with 1,5-I-AEDANS, are shown in Fig When excited at 295 nm, tryptophan residues in the unlabeled protein had a fluorescence emission maximum near 339 nm In the presence of 1,5-I-AEDANS, tryptophan fluorescence intensity was significantly reduced compared with an approximately equal concentration of an unmodified protein The maximum wavelength of tryptophan emission in the labeled mutant W13F was shifted to % 327 nm The addition of cAMP and DNA increased energy transfer from Trp85 residue to the AEDANS moiety The quantum yields of tryptophan fluorescence at 25 °C in buffer A were determined to be 0.09 for mutant CRPW13F alone and 0.094 for mutant CRPW13F in the presence of 200 lM cAMP The quantum yield of the donor increased upon protein–DNA complex formation The change in the observed value of the quantum yield was % 20% A similar result was obtained for CRP– (cAMP)4 Time-resolved fluorescence data FRET measurements Lifetime measurements on the mutant CRPW13F labeled with 1,5-I-AEDANS indicated a decreased lifetime of the tryptophan fluorescence, as expected when energy transfer occurs Figure shows the time-dependent donor decays for the proteins bearing donor alone and those with donor and acceptor In the case of mutant CRPW85A, no energy transfer was observed Lifetime measurements were repeated several times for each species The fluorescence decays were Ó FEBS 2003 1418 A Polit et al (Eur J Biochem 270) Fig Fluorescence emission spectra of unmodified and modified CRPW13F The excitation wavelength was 295 nm and emission was scanned from 310 to 570 nm Measurements were performed at 25 °C in buffer A, pH 8.0 (— Ỉ Ỉ —) Unmodified CRPW13F; (—) modified CRPW13F; (- - -) modified CRPW13F in the presence of 200 lM cAMP; (ỈỈỈỈ) modified CRPW13F bound to DNA in the presence of cAMP analyzed as a multiexponential decay, and for each case the double exponential decay was characterized by lower values of reduced v2 In some cases the triple exponential decays were recorded; however, the pre-exponential factor a3 for these fits was close to zero, and therefore this component of the decay is not included in the calculated average values of fluorescence lifetimes ỈsDỉ and ỈsD The values for average lifetimes of Trp85 in the absence and presence of acceptor and efficiency of energy transfer are presented in Table The transfer efficiency varied between apoprotein and after binding cAMP at micromolar or millimolar concentrations In the absence of a specific ligand, CRPW13F showed an efficiency of transfer of 24.2 ± 8.1% The addition of cAMP had a significant effect on energy transfer The value for CRPW13F with two cAMP molecules bound to anticAMP-binding sites was considerably higher (72.3 ± 2.5%) than for apoprotein The result determined for the mutant W13F in the presence of mM cAMP was similar to the above value, averaging 74.3 ± 2.4% Energy transfer in CRPW13F bound to the specific fragments of DNA was also measured and displayed similar values of efficiency for each complex examined (% 62%) The energy-transfer efficiency values were used to calculate the average distance between the donor and the acceptor To approximate the distance between the Trp85–Cys178 pair, we assumed that the Forster distance ă for the tryptophanIAEDANS pair was 22 A [26,27] The estimated distances are shown in Table There was a significant difference between the calculated distance in apo-CRPW13F and CRPW13F complexed with cAMP and DNA the anisotropy decays of Trp13 and Trp85 could be described by one exponent with single rotational correlation time Addition of the second exponent did not significantly alter the goodness of fit The v2 value obtained with the single-exponential and double-exponential analysis indicates that the two-component analysis is not significantly better than the one-component analysis Also the distribution of the residuals did not improve on the addition of the second component (Fig 6B,C) However, the data obtained for the single exponential analysis suggest the presence of an additional segmental mobility This is evident from apparent time-zero anisotropy r(0), which is lower than the fundamental anisotropy r0 of tryptophan at this excitation wavelength [28] For both tryptophan residues, the initial anisotropy was in the range 0.22–0.23 (Table 2) It indicates that anisotropy decay contains a fast component which cannot be resolved with our device The anisotropy decay parameters for various CRP species are reported in Table The mean ± SD value of rotational correlation times determined from the study of fluorescence anisotropy decays of Trp85 in the absence of specific ligand was 20.5 ± 2.4 ns The value for Trp13 was considerably lower, averaging 15.3 ± 1.8 ns The addition of 100 lM cAMP probably did not affect the rotational correlation time of CRPW13F The uncertainty of this value was too large to ascertain any changes in the CRP dynamic on cAMP binding Trp13 exhibited different behavior The rotational correlation time determined from the study of fluorescence anisotropy decays of Trp13 in the presence of cAMP decreased to 10.45 ± 3.0 ns Time-domain anisotropy data The fluorescence anisotropy decays of Trp13 and Trp85 were measured to determine the changes in the rotational diffusion of CRP after cAMP binding The anisotropy decay of Trp13 in the CRP complexed with two cAMP molecules is shown in Fig 6A Analysis of the anisotropy decays was carried out according to Eqn (8) with an increasing number of exponents, until the fit no longer improved In all cases, Discussion cAMP binding to CRP has been studied using a variety of methods, which have shown that the ligand binding mediates changes in the protein conformation It is believed that these changes allow the protein molecule to switch from the low-affinity and nonspecific DNA-binding state to the state characterized by high affinity and sequence specificity Ó FEBS 2003 Conformational changes induced by cAMP and DNA binding to CRP (Eur J Biochem 270) 1419 Fig Trp85 fluorescence intensity decays for mutant CRPW13F without and with 1,5-I-AEDANS covalently attached to Cys178 The dark grey dotted 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 of the acceptor (DA) The black solid lines and weighted residuals (lower panels) are for the best triple exponential fits Experiments were performed in buffer A at 20 °C for the DNA promoter [2] As the X-ray crystal structure of apo-CRP has not yet been resolved, it is believed that the binding of cAMP, which leads to a switch to active protein conformation, involves subunit realignment and hinge reorientation between the protein domains [2,29] For a long time, it has been a paradigm that CRP undergoes a cAMP concentration-dependent transition between three conformations: apo-CRP, CRP–(cAMP)1 and CRP– (cAMP)2, and each conformer possesses a unique structure and activity [2] The reinterpretation of this paradigm was proposed by Passner & Steitz [6] on the basis of the crystal structure of the CRP–cAMP complex, and it has recently been supported by NMR [7] and isothermal titration calorimetry [8] studies in solution NMR experiments have shown that CRP possesses two anti-cAMP-binding sites in each monomer, and the next two syn-cAMP sites are formed by an allosteric conformational change in the protein on biding of two anti-AMP at the N-terminal Ó FEBS 2003 1420 A Polit et al (Eur J Biochem 270) Table Summary of energy transfer measurements In the CRPW13F–(cAMP)2 complex, the concentration of cAMP was 200 lM, whereas in the case of CRPW13F–(cAMP)4 the concentration of cAMP was mM The molar ratio CRP to DNA in the protein–DNA complex was : Species ỈsDỉ (ns) ỈsD (ns) CRPW13F CRPW13F–(cAMP)2 CRPW13F–(cAMP)4 CRPW13F–ICAP CRPW13F–lac CRPW13F–gal 5.83 5.59 5.99 5.55 5.85 5.85 4.42 1.55 1.54 2.10 2.23 2.17 24.2 72.3 74.3 62.2 61.9 62.9 ˚ ỈRỉ (A) Ỉ (%) ± ± ± ± ± ± 0.50 0.31 0.42 0.25 0.16 0.12 domain [7] The isothermal titration calorimetry measurements demonstrated that, at low cAMP concentration, there are two identical interactive high-affinity sites for cAMP and at least one low-affinity cAMP-binding site at high concentration of the ligand [8] The idea of the four cAMP-binding sites in CRP, for its anti conformation (at low concentration of the ligand) and the next two binding it in syn conformation (at high cAMP concentration) has been used to describe the results of fast kinetic studies [15] and investigations with dynamic light scattering and time-resolved fluorescence anisotropy measurements [30] In these studies, we have shown that the binding of cAMP in anti conformation in the N-terminal domain of CRP leads to the conformational changes in the helix–turn–helix motif of the C-terminal domain, responsible for the interaction with DNA, as well as to the changes in the global hydrodynamic structure of CRP The saturation of the low-affinity sites with cAMP in syn conformation of cAMP results in the changes in the microenvironment of the Trp85 residue, localized in the N-terminal domain of the protein, without further substantial changes in the global hydrodynamic structure [30] The results presented in this report provide further evidence for conformational changes induced by cAMP binding to the anti-cAMP-binding sites of CRP, which in turn trigger specific pathways of signal transmission from the cyclic nucleotide-binding domain to the DNA-binding domain of the protein We have used single tryptophancontaining mutants of CRP The mutations were localized in the N-terminal domain at position 85 or 13 in order to follow conformational changes in their microenvironment on binding of cAMP, both in anti- and syn- conformation We have also used AEDANS for fluorescent labeling of Cys178, located at the turn of the helix–turn–helix motif, in order to detect FRET between Trp85 and the label We have shown that binding of cAMP at a concentration of 200 lM to anti-cAMP-binding sites results in % 13% decrease in the Trp13 fluorescence intensity along with the blue shift in its maximum of emission by 2.5 nm Probable candidates for quenching residues in CRP are Thr10, Asn109 and His17, which are located within a distance up to ˚ A, as has been determined from the X-ray crystal structure of the CRP–cAMP complex (PDB code 1G6N) [29] The observed changes in the microenvironment of Trp13 on filling of the high-affinity sites are also supported by the time-resolved anisotropy measurements Binding of the ligand to anti-cAMP-binding sites results in a decrease in rotational correlation time by % ns, from the value of 15.3 ns, detected for apo-CRP, to the value of 10.4 ns for CRP–(cAMP)2 complex The decrease in rotational time ± ± ± ± ± ± 0.28 0.11 0.10 0.06 0.06 0.25 ± ± ± ± ± ± 8.1 2.5 2.4 2.0 1.5 4.3 26.6 18.7 18.4 20.2 20.3 20.1 ± ± ± ± ± ± 3.9 0.8 0.8 0.6 0.5 1.2 indicates an increase in the mobility of helix A of the protein As Trp13 is located in the vicinity of the activation region AR2 of the protein [1], which is responsible for the activation of the second class of E coli promoters such as gal P1, one can speculate that this conformational change may play an important role in a signal transmission in the protein molecule, which in turn may allow CRP to adopt a conformation appropriate for the interaction with the aNTD domain of RNA polymerase in the transcription complex On the other hand, Trp13 of CRP directly interacts with another gene regulatory protein, CytR [31], and the observed conformational changes in Trp13 microenvironments on cAMP binding to anti-cAMP sites may play a significant role in the CRP–CytR–DNA complex In contrast with Trp13 of CRP, binding of cAMP to anticAMP-binding sites does not lead to a significant change in fluorescence intensity of Trp85, and only a % 3.4% increase in the intensity has been observed at 200 lM cAMP However, cAMP binding to the syn-cAMP-binding sites at concentration of the ligand of mM causes a % 6% increase in its fluorescence intensity, which indicates that this residue is sensitive to cAMP binding to low-affinity sites The rotational correlation time of Trp85 in apo-CRP of 20.5 ns indicates that this residue is immobilized within the N-terminal domain of the protein and exhibits motion characteristic of the whole protein (within experimental error), while the respective value for the AEDANS-labeled apo-CRP has been estimated at 23.3 ns [30] Binding of cAMP to anti-cAMP-binding sites increases the rotational correlation time to 22 ns for the CRP–(cAMP)2 complex, which is much lower than the correlation time of 30 ns determined for this complex using CRP labeled at Cys178 with the AEDANS fluorescent probe [30] These discrepancies can probably be explained by the fact that the average fluorescence lifetime of Trp85, % 5.6 ns in the case of the CRP–(cAMP)2 complex, is too short to allow observations of the longer rotational correlation times The allosteric activation of CRP involves conformational changes in the N-terminal domain of the protein and leads to changes in the CRP molecule, enabling it to recognize the specific DNA sequence [1,2] As the crystal structure of apoCRP has not yet been established, it was suggested that cAMP binding may cause reorientation of the coiled-coil C helices, consequently altering the relative position of the protein dimer subunits [29] These authors also suggested that, in the absence of cAMP in apo-CRP, some b strands of the N-terminal domain of the protein may collapse into the cAMP-binding pocket, causing reorientation of the smaller domain in relation to the larger one, and bringing these domains closer together This suggestion has been Ó FEBS 2003 Conformational changes induced by cAMP and DNA binding to CRP (Eur J Biochem 270) 1421 Fig Time-domain fluorescence anisotropy decay of Trp13 in the presence of 100 lM cAMP The solid line corresponds to the best single exponential fit of the data (dotted curve) according to Eqn (8) The grey cross-haired curve represents the lamp profile The plots of the residuals for the 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 Table Parameters of Trp13 and Trp85 anisotropy decays in the presence and absence of cAMP Species h (ns) CRPW13F CRPW85A CRPW13F–(cAMP)2 CRPW85A–(cAMP)2 20.5 15.3 22.1 10.45 v2 r(0) ± ± ± ± 2.4 1.8 6.9 3.0 0.23 0.21 0.23 0.23 ± ± ± ± 0.02 0.02 0.03 0.03 1.222 1.055 1.036 1.146 supported recently by NMR studies [7]; these authors argue that binding in solution of two cAMP molecules to highaffinity anti-cAMP-binding sites at the N-terminal domain causes the C-terminal domain to shift further to the N-terminal domain of CRP To confirm this suggestion, we used FRET to detect the distance between the C-terminal and N-terminal domains of CRP on binding of cAMP in anti as well as syn conformation to the protein For this purpose, we used time-resolved fluorescence lifetime measurements Ó FEBS 2003 1422 A Polit et al (Eur J Biochem 270) using single tryptophan-containing mutants of CRP Fluorescence energy transfer could be detected between Trp85, localized close to the cAMP-binding pocket of the N-terminal domain, and Cys178, fluorescently labeled by AEDANS, localized in the helix–turn–helix motif of the C-terminal domain of the protein The lifetimes obtained for the fluorescence donor, Trp85, indicate that binding of cAMP to anti-cAMP-binding sites leads to a dramatic increase in FRET efficiency This observation clearly shows a decrease in the average distance between the two domains of CRP on cAMP binding If one assumes the Forster distance, ă R0, for the pair donor–acceptor such as tryptophan– ˚ AEDANS to be 22 A [26,27], the distance between Trp85 and Cys178-AEDANS in the apo-CRP can be calculated to ˚ ˚ ˚ be 26.6 A This distance decreases by about A to 18.7 A on binding of cAMP to the anti-cAMP-binding sites of the protein The distance between the sulfur atom of Cys178 and the C9–C10 bond of the indole ring of Trp85, derived from the crystal structure of CRP–(cAMP)2 (PDB code 1G6N) is ˚ ˚ 18.9 A and 21.9 A for the subunit present in the ÔclosedÕ and ÔopenÕ conformation, respectively [29] The structural asymmetry of the CRP–(cAMP)2 complex resulting from conformational differences between subunits has been questioned [32], and from molecular dynamics simulation, it has been predicted that, in solution, both subunits of CRP adopt a ÔclosedÕ conformation If this is so, the distance (equal ˚ to 18.7 A), determined in this work by FRET, is in good ˚ agreement with the value of 18.9 A predicted for the ÔclosedÕ conformation This supports experimentally the dynamic simulation studies [32] and indicates that, in solution, both of the protein subunits exist in ÔclosedÕ conformation in the CRP–(cAMP)2 complex Because a variety of spectroscopic effects, at least in theory, could influence energy transfer efficiency, one can argue that the good agreement determined for the distance between Cys178 and Trp85 residues in CRP–(cAMP)2 may also result from the assumed value of Forster distance R0 ă However, as binding of cAMP at a concentration of 200 lM to CRP leads only to a % 4.3% increase in the fluorescence quantum yields from the value of 0.09 for apo-CRP to the value of 0.094 for the CRP–(cAMP)2 complex, and no substantial changes in the shape of the emission spectra of the donor have been observed, this justifies the lack of the alteration of the Forster distance between apo and holo ă forms of the protein As the AEDANS label attached to the Cys178 enjoys local freedom of movement, in both apoCRP and the CRP–(cAMP)2 complex [30], the distance obtained from the crystal structure between the sulfur atom of Cys178 and the indole ring of Trp85 seems to be realistic We have also tried to measure fluorescence energy transfer between Trp13 and Cys178-AEDANS; however, we have not detected any energy transfer in either the apo- or holoform This could be because of the distance between the two ˚ residues, which is about 45 A, as can be calculated from the crystal structure of CRP–(cAMP)2 [29] Binding of cAMP in syn conformation to the lowaffinity binding sites in the CRP–(cAMP)4 complex leads to only a small increase in the efficiency of energy ˚ transfer, which, with an assumed R0 value of 22 A, corresponds to the small decrease in average distance between the N-terminal and C-terminal domains of CRP, ˚ estimated at 18.4 A However, the fluorescence quantum yield of the Trp85 donor increases by % 20% at a concentration of cAMP of mM from the value characteristic of apo-CRP, which in turn may be responsible for this very small change We have also measured the distance between the two domains of CRP in the complexes with DNA containing various sequences, such as lac and gal promoters and with the symmetric sequence ICAP For each CRP–DNA complex, the increase in the distance between the two CRP domains ˚ has been observed with the average distance of 20.2 A This value is in good agreement with the value of ˚ 20.7 A, calculated from the crystal structure of the CRP– DNA complex [33] The present results show that the binding of anti-cAMP in the CRP–(cAMP)2 complex results in the movement of ˚ the C-terminal domain of CRP by % A towards the N-terminal domain, which in consequence leads to rearrangement of DNA-binding domains and cAMPbinding domains of the protein This finding clarifies the suggestion derived from the NMR measurements [7] that the C-terminus is closer to the N-terminal domain in apoCRP than in cAMP-bound CRP Binding of cAMP to anti-cAMP-binding sites leads to an increase in the structural dynamic motion around Trp13, which is close to the activation region AR2, responsible for the interaction of CRP with the a subunit of RNA polymerase The changes in the CRP dynamics on cAMP binding have recently been observed by the hydrogen exchange method [34] In that paper, it was shown that binding of the ligand to the protein causes the C-terminal domain of CRP to become more flexible, in contrast with the Nterminal domain which is shifted to a less dynamic conformation Our results extend this observation and suggest that the binding of cAMP to anti-cAMP-binding sites of CRP leads to the increase in the structural dynamic motion of at least Trp13, which is located in the N-terminal domain of the protein Acknowledgements We are grateful to Dr S Garges for supplying us with the plasmid for production of CRP This work was supported by grant no P04A 031 16 from the State Committee for Scientific Research References Busby, S & Ebright, R (1999) Transcription activation by Catabolite Activator Protein (CAP) J Mol Biol 293, 199–213 Harman, J.G (2001) Allosteric regulation of the cAMP receptor protein Biochim Biophys Acta 2, 1–17 de Crombrugghe, B., Busby, S & Buc, H (1984) Cyclic AMP receptor protein: role in transcription activation Science 224, 831–838 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 Parkinson, G., Wilson, C., Gunasekera, A., Ebright, Y.W., Ebright, R.E & Berman, H (1996) Structure of the CAP–DNA ˚ complex at 2.5 A resolution: a complete picture of the protein– DNA interface J Mol Biol 260, 395–408 Passner, J.M & Steitz, T.A (1997) The structure of a CAP–DNA complex having two cAMP molecules bound to each monomer Proc Natl Acad Sci USA 94, 2843–2847 Ó FEBS 2003 Conformational changes induced by cAMP and DNA binding to CRP (Eur J Biochem 270) 1423 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 Lin, S.-H & Lee, J.C (2002) Communications between the highaffinity cyclic nucleotide binding sites in E coli cyclic AMP receptor protein: effect of single site mutations Biochemistry 41, 11857–11867 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 Proc Natl Acad Sci USA 76, 5090–5094 11 Mukhopadhyay, J., Sur, R & Parrack, P (1999) Functional 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 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 275, 8480–8486 16 Merck Inc (1976) The Merck Index 9th edn Merck Inc., Rahway, NJ, USA 17 Takahashi, M., Blazy, B & Baudras, A (1980) An equilibrium 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 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) amino]ethyl]-5naphtylamine-1-sulfonate 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 Ebright, R.H., Le Grice, S.F., Miller, J.P & Krakow, J.S (1985) Analogs of cyclic AMP that elicit the biochemically defined conformational change in catabolite activator protein (CAP) but not stimulate binding to DNA J Mol Biol 182, 91–107 26 Fairclough, R.H & Cantor, C.R (1978) The use of singlet-singlet energy transfer to study macromolecular assemblies Methods Enzymol 48, 347–379 27 Selvin, P.R (1995) Fluorescence resonance energy transfer Methods Enzymol 248, 300–334 28 Lakowicz, J.R., Maliwal, B.P., Cherek, H & Balter, A (1983) Rotational 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 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 32 Garcia, A.E & Harman, J.G (1996) 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 dynamics changes in Escherichia coli cyclic AMP receptor protein Biochemistry 41, 6660–6667  ... 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. .. calculated to ˚ ˚ ˚ be 26.6 A This distance decreases by about A to 18.7 A on binding of cAMP to the anti-cAMP -binding sites of the protein The distance between the sulfur atom of Cys178 and the... nucleotide binding sites in E coli cyclic AMP receptor protein: effect of single site mutations Biochemistry 41, 11857–11867 Heyduk, T & Lee, J.S (1989) Escherichia coli cAMP receptor protein: evidence