Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli Magdalena Tworzydło, Agnieszka Polit, Jan Mikołajczak and Zygmunt Wasylewski Department of Physical Biochemistry, Faculty of Biotechnology, Jagiellonian University, Krako ´ w, Poland Cyclic AMP receptor protein (CRP), allosterically activated by cAMP, is a multipotent transcription regulating protein engaged in the control of more then 100 genes in Escherichia coli [1,2]. The protein is a homodimer. Each subunit consists of 209 amino acid residues folded into two distinct domains. The N-terminal domain, composed of amino acid residues 1–133, contains a cAMP-binding pocket that binds the cAMP in the anti conformation. The N-terminal domain is coupled with the C-terminal domain by a flexible hinge region made up of residues 134–138. The smaller, C-terminal domain possesses amino acid residues 139–209 and contains the helix-turn-helix (HTH) motif. The crystal structure of the CRP–DNA complex revealed the existence of a second site between the hinge and the turn of the HTH where cAMP is bound in the syn conformation [3]. Upon cAMP binding in the anti conformation, CRP under- goes allosteric conformational changes that enable the protein to recognize specific DNA sequences [2,4]. Therefore, it has been suggested that CRP can exist in solution in at least three conformational states, Keywords cAMP receptor protein (CRP); CRP–DNA interactions; fluorescence quenching; FRET, fast kinetics Correspondence Z. Wasylewski, Department of Physical Biochemistry, Faculty of Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30–387 Krako ´ w, Poland Fax: +48 12 66 46 902 Tel: +48 12 66 46 122 E-mail: wasylewski@mol.uj.edu.pl (Received 29 July 2004, revised 22 November 2004, accepted 21 December 2004) doi:10.1111/j.1742-4658.2005.04540.x Cyclic AMP receptor protein (CRP) regulates the expression of more then 100 genes in Escherichia coli. It is known that the allosteric activation of CRP by cAMP involves a long-distance signal transmission from the N-ter- minal cAMP-binding domain to the C-terminal domain of CRP responsible for the interactions with specific sequences of DNA. In this report we have used a CRP mutant containing a single Trp13 located in the N-terminal domain of the protein. We applied the iodide and acrylamide fluorescence quenching method in order to study how different DNA sequences and cAMP binding induce the conformational changes in the CRP molecule. The results presented provide evidence for the occurrence of a long- distance conformational signal transduction within the protein from the C-terminal DNA-binding domain to the N-terminal domain of CRP. This conformational signal transmission depends on the promoter sequence. We also used the stopped-flow and Fo ¨ rster resonance energy transfer between labeled Cys178 of CRP and fluorescently labeled DNA sequences to study the kinetics of DNA–CRP interactions. The results thus obtained lead to the conclusion that CRP can exist in several conformational states and that their distribution is affected by binding of both the cAMP and of specific DNA sequences. Abbreviations CRP, cyclic AMP receptor protein; CRP–AEDANS, CRP covalently labeled with 1,5-I-AEDANS attached to Cys178; apo–CRP, unligated CRP; FRET, Fo ¨ rster resonance energy transfer; FQRS, fluorescence-quenching-resolved spectra; galF, a fragment of DNA sequence recognized by CRP in the galP1 promoter covalently labeled with fluorescein at the 5¢ end; HTH, helix-turn-helix; lacF, a fragment of DNA sequence recognized by CRP in the lacP1 promoter covalently labeled with fluorescein at the 5¢ end; ICAPF, consensus DNA sequence recognized by CRP covalently labeled with fluorescein at the 5¢-end; wt, wild type. FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS 1103 i.e. free CRP, CRP–(cAMP) 2 and CRP–(cAMP) 4 .In the presence of % 100 lm cAMP, the protein becomes activated by the formation of a CRP–(cAMP) 2 com- plex and it is then able to recognize and bind specific DNA sequences and stimulate transcription [5]. Unfortunately, the crystal structure of unligated CRP has not yet been established, which makes a simple comparison between the two forms of the protein impossible. However, it has been suggested from the crystal structure studies that the cAMP-induced allo- steric transition may involve a change in relative ori- entation of the subunits and a change in orientation of the DNA-binding domain relative to the cAMP- binding domain [6]. Indeed, our Fo ¨ rster resonance energy transfer (FRET) measurements show that the binding of anticAMP in the CRP–(cAMP) 2 complex results in a movement of the C-terminal domain of CRP by % 8A ˚ towards the N-terminal domain [7]. As in the CRP–(cAMP) 2 complex the anticAMP is buried within the N-terminal domain of the protein located at least 10 A ˚ away from the hinge region, the allosteric activation of CRP must involve a long- distance signal transmission within the protein. Recent studies [8] suggest that this long-distance communica- tion between the two CRP domains and subunits involves the Asp138 residue, located in the CRP hinge region, which represents part of the signal transduc- tion network. Depending on the location of the CRP-binding site on the DNA promoter and the mechanism of CRP– RNA polymerase interaction, the simple CRP-depend- ent promoters are divided into two classes [1]. Class I promoters, such as lacP1, are characterized by the location of the CRP-binding site centred at position )61.5. In the case of class II promoters, such as galP1, the CRP-binding site is located at position )41.5. The activation of the transcription process requires the interaction between the RNA polymerase a subunit C-terminal domain and the CRP-activating region, AR1 [9]. The class II promoter requires the interaction with both the AR1 activation region of CRP and the activation region of AR2, located in the CRP N-terminal domain [10]. Each CRP subunit contains two tryptophan residues at positions 13 and 85 (Fig. 1), both located in the protein’s N-terminal domain [11]. Trp85 is located near the anticAMP-binding site and Trp13 is situated close to the activation region, AR2, of CRP. Using single tryptophan-containing mutants, we have recently shown that the binding of cAMP in the CRP–(cAMP) 2 complex alters the surroundings of Trp13, whereas its binding in the CRP–(cAMP) 4 complex leads to changes in the Trp85 microenvironment [7]. We present evidence that CRP binding to the different DNA sequences leads to long-distance conformational signal transmission from the C-terminal domain to the N-terminal domain of the protein. Furthermore, we present the kinetics of DNA–CRP interactions, as determined by using FRET measurements, between labeled Cys178 of CRP and fluorescently labeled DNA sequences (Fig. 1). The mechanism of the cAMP-induced long-distance structural communication within the CRP remains an important part of our understanding of the mechan- ism underlying the transcription-regulating activity of this protein. However, it is an open question as to how the binding of the CRP–(cAMP) 2 complex to different specific promoter DNA sequences can trigger the conformational changes in the protein that may consequently lead to changes in the interactions between the activator and other participants of the transcription machinery. Does it involve a conforma- tional signal transmission from the C-terminal domain of CRP through the hinge region to the N-terminal domain? We believe that elucidation of the signal transduction pathway from the different DNA sequences to the activation regions in CRP may pro- vide a structural paradigm for understanding the tran- scription activation process. Therefore, we suggest that the CRP does not act by the simple ‘recruitment’ mechanism in transcription machinery, as has been suggested recently [12], but behaves as a very dynamic entity. Fig. 1. Structure of the cyclic AMP receptor protein (CRP) dimer complexed with DNA. The locations of tryptophan residues are marked in red, the location of the Cys178 residue is indicated in yellow and fluorescein is shown in green. The figure was generated by WEBLAB VIEWERPRO (version 3.7) using atomic coordinates for the cAMP–CRP–DNA complex [44]. The coordinates were obtained from the Brookhaven Protein Data Bank (accession code 1CGP). CRP conformational changes induced by DNA and cAMP M. Tworzydło et al. 1104 FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS Results Steady-state fluorescence quenching studies The fluorescence quenching studies with iodide and acrylamide were performed in 20 mm Tris⁄ HCl buffer, pH 7.9, containing 0.1 m NaCl and 0.1 mm EDTA. In measurements involving the protein–ligand complex, the final concentration of cAMP was 100 lm. In all cases, the excitation wavelength was 295 nm, so it can be assumed that the fluorescence emission observed was only from tryptophan residues. A typical Stern–Volmer plot of fluorescence quench- ing of the single tryptophan of the CRPW85A mutant is shown in Fig. 2. The downward curvature of the plot indicates the presence of two or more emitting components which differ in a Stern–Volmer quenching constant, K SV . The fluorescence quenching data were analyzed according to Eqn (3), by using a nonlinear least-squares procedure. The analysis was conducted for all the quenching data, i.e. for about 40 different emission spectra. Judging by the calculated v 2 value and the residual distribution, the phenomenon can be described by a two-component model in which one component in the protein is more available for the quencher and characterized by K SV1 ¼ 9.61 m )1 and an f 1 of % 0.55, while the other component is less accessible to the iodide with K SV2 ¼ 1.69 m )1 and f 2 ¼ 0.45. The best theoretical-fit line calculated for the given emission wavelength is shown in Fig. 2A. Similar results were obtained for the CRPW85A– (cAMP) 2 complex. The Stern–Volmer plot also curved down (data not shown). The binding of cAMP resulted in a small increase of the K SV1 value from 9.61 m )1 to 10.08 m )1 and the more visible increase of the K SV2 value from 1.69 m )1 to 2.85 m )1 . When acrylamide was used as a quencher, the Stern–Volmer plots of CRPW85A and its complex with cAMP showed a small upward curvature indica- ting that a static quenching mechanism is involved (Fig. 2B). For both species, the best fits were obtained for a model in which one component is accessible to the nonionic quencher. For CRPW85A, the acrylamide Stern–Volmer constant is equal to 5.76 m )1 , while for the cAMP complex, K SV ¼ 6.62 m )1 , and the values of a static quenching constant, V, are 0.84 m )1 and 0.27 m )1 , respectively. The fitting parameters for iodide and acrylamide quenching are given in Table 1. Figure 3 shows, for the first time, the spectra of CRP, containing a single Trp13 residue, resolved into components by using the fluorescence-quenching- resolved spectra (FQRS) method, using iodide as a quencher. The component characterized by a higher Stern–Volmer constant (9.61 m )1 ) was found to exhibit a maximum at 350 nm and to account for 55% of the fluorescence emission. The second component, charac- terized by the average K SV ¼ 1.69 m )1 , is responsible for % 45% of the total emission and has a maximum at 338 nm. Fig. 2. (A) Typical Stern–Volmer plot for iodide quenching of CRPW85A ( ). The solid line represents the best fit with the fol- lowing parameters: K SV1 ¼ 9.11 M )1 , f 1 ¼ 0.48, K SV2 ¼ 2.89 M )1 , f 2 ¼ 0.40. (B) Typical Stern–Volmer plots for acrylamide quenching of CRPW85A ( ) and of CRPW85A–(cAMP) 2 (h). The solid lines represent the best fits with the following parameters: CRPW85A, K SV ¼ 5.64 M )1 , V ¼ 0.74 M )1 , f ¼ 1; CRPW85A–(cAMP) 2 , K SV ¼ 6.45 M )1 , V ¼ 0.22 M )1 , f ¼ 1. The excitation was at 295 nm and the emission at 340 nm. M. Tworzydło et al. CRP conformational changes induced by DNA and cAMP FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS 1105 FQRS spectra of CRPW85A with cAMP are repre- sented in Fig. 4. The binding of the ligand results in a blue shift of the total spectrum maximum from about 342 nm to 340 nm. The more quenchable component exhibits a k max at 344 nm, whereas the maximum of the less quenchable component remains unchanged at 338 nm. The maxima of the resolved spectra and their relative intensities, measured as the areas under each of the resolved spectra, are given in Table 1. Analogous measurements were performed for CRP– DNA complexes. Figure 5A,B shows typical Stern– Volmer plots obtained for iodide and acrylamide quenching of the CRPW85A mutant bound to ICAP, lac and gal sequences. For all three DNA fragments, the Stern–Volmer plots of fluorescence quenching by iodide exhibit a downward curvature, and the best fits were obtained with a two-component model in which one component is quenchable and the second remains inaccessible for the quencher. In order to prove that the downward curvature was not a result of the ionic strength chan- ges when iodide was added, the titration of the CRPW85A–DNA complexes with KCl was performed and it did not lead to any substantial changes in the fluorescence emission of the complexes. The high- est Stern–Volmer constant, amounting to 7.45 m )1 , characterizes the CRPW85A–ICAP complex. For CRPW85A–lac, the value of K SV1 is 5.54 m )1 , and for CRPW85A–gal, the value of K SV2 is 5.02 m )1 . The quenched components account for % 78–81% of the total fluorescence emission. When acrylamide was used for quenching, the Stern–Volmer plots for two complexes of CRPW85A, with ICAP and lac sequences, were found to be linear so the model with one totally quenched com- ponent was used for calculations. The dynamic quenching constant values for these two species were 6.35 and 6.15 m )1 , respectively. Only for the CRPW85A–gal complex did the upward curvature appear, indicating the presence of static quenching, characterized by the constant V ¼ 1.35 m )1 . The K SV for the CRPW85A–gal complex was lower than for the complexes with the ICAP and lac sequences and equaled 5.53 m )1 . The total fluorescence emission of all three CRPW85A–DNA complexes had maxima at the same wavelength as the CRPW85A–(cAMP) 2 complex (Figs 6, 7 and 8), i.e. at 340 nm. The resolved spectra which correspond to the unquenchable components have maxima at around 338 nm, while the maxima of quenchable components are located at around 344 nm. The detailed parameters of the resolved spectra of the CRPW85A–DNA complexes, with iodide used as a quencher, are presented in Table 1. Time-resolved fluorescence data Fluorescence lifetime measurements of the CRPW85A mutant and its complexes with cAMP and DNA were conducted using an excitation wavelength equal to 295 nm. Phase and modulation were analyzed by using single- and double-exponential decay models. The bet- ter fits, i.e. of lower values of the reduced v 2 , were obtained for a double-exponential model. The values of mean fluorescence lifetimes, defined as s m ¼ Sf i s, are presented in Table 2. Table 1. Fluorescence quenching parameters for CRPW85A, CRPW85A–(cAMP) 2 and CRPW85A–DNA complexes. Iodide and acrylamide quenching studies were performed in Tris buffer, pH 7.9 at 20 °C. In the experiments with CRPW85A complexed to cAMP and DNA, the concentration of cAMP was 100 l M. Quenching data were fitted to either a one- or a two-component model (Eqn 1). The presented parame- ters were obtained for the model characterized by minimum values of reduced v 2 . K SV and V are average values calculated for the wave- length range between 330 and 370 nm. The error did not exceed 5%. FQRS, fluorescence-quenching-resolved spectra. Species K SV1 (M )1 ) K SV2 (M )1 ) V (M )1 ) f 1 FQRS k maks1 (nm) k maks2 (nm) Iodide quenching CRPW85A 9.61 1.69 – 0.55 350 338 CRPW85A–(cAMP) 2 10.08 2.85 – 0.57 344 338 CRPW85A–ICAP 7.45 0.00 – 0.80 345 338 CRPW85A–lac 5.54 0.00 – 0.78 346 340 CRPW85A–gal 5.02 0.00 – 0.81 343 337 Acrylamide quenching CRPW85A 5.76 – 0.84 1.00 – – CRPW85A–(cAMP) 2 6.62 – 0.27 1.00 – – CRPW85A–ICAP 6.35 – – 1.00 – – CRPW85A–lac 6.15 – – 1.00 – – CRPW85A–gal 5.53 – 1.33 1.00 – – CRP conformational changes induced by DNA and cAMP M. Tworzydło et al. 1106 FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS Kinetics of DNA binding to CRP A FRET has been used to study the kinetics of CRP– DNA interactions. The fluorescence characteristics of CRP-conjugated IAEDANS, with an excitation at 340 nm and a maximum emission at 480 nm, suggest that it can be used as a donor fluorophore. Oligonucleo- tides covalently labeled with fluorescein were used as acceptors. The application of the FRET method allowed us to obtain more information about the binding process between protein and DNA. One of the advantages is the possibility of determining the kinetics of the associ- ation by monitoring the time course of the FRET effect. Using fluorescein-labeled DNA as the acceptor, we observed a small increase in acceptor fluorescence but a significant decrease in IAEDANS emission. Quenching of the IAEDANS fluorescence intensities is not solely governed by Fo ¨ rster nonradiative energy transfer in the CRP–DNA complex, but also by the DNA itself. The addition of unlabeled DNA to CRP– AEDANS significantly decreased the fluorescence intensities of the dye (data not shown) and therefore we decided to use the acceptor fluorescence to monitor the CRP–DNA interaction in the FRET kinetic measure- ments. Mixing an IAEDANS-labeled CRP with a fluo- rescein-labeled oligonucleotide resulted in an increase of % 7% in the acceptor fluorescence at the donor exci- tation wavelength, reaching a plateau at % 0.3 s. For all DNA sequences and CRP concentrations, the kinetic traces could be fitted well by a single-expo- nential curve. The plots of the inverse time constant (k obs ) are linear (Fig. 9) and the values of k off and the association-rate parameter, k on , listed in Table 3, were determined as the intercept and the slope that are valid for a single-step bimolecular association: Fig. 4. Fluorescence-quenching-resolved spectra (FQRS) of CRPW85A–(cAMP) 2 with excitation at 295 nm. Iodide was used as a quencher. The upper panel represents a plot of Stern–Volmer constants as a function of the emission wavelength. The lower panel shows the FQRS: ( ) the total emission spectrum with a maximum at about 340 nm; (h ) the more quenchable component with a maximum at % 344 nm, characterized by an average value of K SV1 ¼ 10.08 M )1 and a fraction f 1 ¼ 0.57; ( ) the less quenchable component with the maximum at % 338 nm, characterized by an average value of K SV2 ¼ 2.85 M )1 and a fraction f 2 ¼ 0.43. Fig. 3. Fluorescence-quenching-resolved spectra (FQRS) of CRPW85A with excitation at 295 nm. Iodide was used as a quen- cher. The upper panel represents a plot of Stern–Volmer constants as a function of the emission wavelength. The lower panel shows the FQRS spectra: ( ) the total emission spectrum with a maxi- mum at about 342 nm; ( ) the more quenchable component with a maximum at about 350 nm, characterized by an average value of K SV1 ¼ 9.61 M )1 and a fraction f 1 ¼ 0.55; and ( ) the less quencha- ble component with a maximum at about 338 nm, characterized by an average value of K SV2 ¼ 1.69 M )1 and a fraction f 2 ¼ 0.45. M. Tworzydło et al. CRP conformational changes induced by DNA and cAMP FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS 1107 CRPÀðcAMPÞ 2 þ DNA À! k on k off DNAÀCRPÀðcAMPÞ 2 and which k obs ¼ k off þ k on ½CRPÀAEDANSð1Þ with the total concentration used of IAEDANS attached to CRP denoted as [CRP–AEDANS]. An equilibrium binding constant can be calculated from the ratio of the rate constants k on and k off as follows: K a ¼ k on k off ð2Þ Association constants (K a ) of CRP with the three investigated sequences of DNA – lacF, galF and ICAPF – are summarized in Table 3. Discussion The molecular mechanism of signal transduction within CRP upon binding of the allosteric inductor to CRP high-affinity binding sites involves a sequence of protein conformational changes, which shift the protein from a low-affinity nonspecific DNA-binding protein to a state of the protein that binds DNA with Fig. 5. (A) Typical Stern–Volmer plots for iodide quenching of CRPW85A complexes with DNA. The solid lines represent the best fits with the following parameters: (r) CRPW85A–ICAP, K SV1 ¼ 6.57 M )1 , f 1 ¼ 0.80; (d) CRPW85A–lac, K SV1 ¼ 5.46 M )1 , f 1 ¼ 0.78; and (,) CRPW85A–gal, K SV1 ¼ 4.63 M )1 , f 1 ¼ 0.81. (B) Typical Stern–Volmer plots for acrylamide quenching of CRPW85A com- plexes with DNA. The solid lines represent the best fits with the following parameters: (e) CRPW85A–ICAP, K SV ¼ 5.92 M )1 , f ¼ 1; (d) CRPW85A–lac, K SV ¼ 5.74 M )1 , f ¼ 1; (.) CRPW85A–gal, K SV ¼ 5.30 M )1 , V ¼ 1.16 M )1 , f ¼ 1. The excitation was at 295 nm and the emission at 340 nm. Fig. 6. Fluorescence-quenching-resolved spectra (FQRS) of CRPW85A–ICAP with excitation at 295 nm. Iodide was used as a quencher. The upper panel represents a plot of Stern–Volmer con- stant as a function of the emission wavelength. The lower panel shows the FQRS: (e) the total emission spectrum with maximum at % 340 nm; (r) the quenchable component with a maximum at % 345 nm, characterized by an average value of K SV1 ¼ 7.45 M )1 and a fraction f 1 ¼ 0.80; and ( ) the unquenchable component with a maximum at %338 nm, characterized by an average value of K SV2 ¼ 0.00 M )1 and a fraction f 2 ¼ 0.20. CRP conformational changes induced by DNA and cAMP M. Tworzydło et al. 1108 FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS high affinity and sequence specificity [2]. A variety of biochemical and biophysical studies [13–16], including our fast-kinetics studies [17,18], as well as steady-state and time-resolved fluorescence [7,19] investigations, have shown that the allosteric mechanism involves sub- unit realignment and hinge reorientation between the domains. Our previous FRET measurements have shown that cAMP binding to the anti sites of CRP shifts the average distance from the C-terminal domain towards the N-terminal domain from 26.6 A ˚ in apo– CRP to 18.7 A ˚ in the CRP–(cAMP) 2 complex [7]. The details of the structural mechanism of CRP activation by a cAMP have not been established because of the lack of an X-ray structure for apo–CRP. However, it may be expected that the binding of an allosteric inductor, cAMP, as well as an interaction of the protein with the specific DNA promoter sequences in solution can lead to changes in the protein activation regions, which in turn allows CRP to interact with the a subunit of RNA polymerase. Recently [7] we have suggested that cAMP binding to anti sites leads to an increase in the structural dynamic motion around the Trp13 residue, which is close to the activation region AR2, responsible for the interaction of CRP with RNA polymerase [10]. The tryptophan residue is widely used as an intrinsic fluorescence probe to observe changes in protein struc- ture [20]. High indole sensitivity to its microenviron- ment in a protein moiety can be used to follow protein structural changes, especially if the complicated emis- sion of tryptophan residues may be resolved into com- ponents. The difficulties in the interpretation of its fluorescence emission result from the dynamics of pro- tein structure and the multiple ground-state conformers, each of which is characterized by distinct tryptophan Fig. 7. Fluorescence-quenching-resolved spectra (FQRS) of CRPW85A–lac with excitation at 295 nm. Iodide was used as a quencher. The upper panel represents a plot of Stern–Volmer con- stant as a function of the emission wavelength. The lower panel shows the FQRS spectra: (s) the total emission spectrum with a maximum at % 340 nm; (d) the quenchable component with a maximum at % 346 nm, characterized by an average value of K SV1 ¼ 5.54 M )1 and a fraction f 1 ¼ 0.78; and ( ) the unquen- chable component with a maximum at % 340 nm, characterized by an average value of K SV2 ¼ 0.00 M )1 and a fraction f 2 ¼ 0.22. Fig. 8. Fluorescence-quenching-resolved spectra (FQRS) of CRPW85A–gal with excitation at 295 nm. Iodide was used as a quencher. The upper panel represents a plot of Stern–Volmer con- stant as a function of the emission wavelength. The lower panel shows the FQRS: (,) the total emission spectrum with a maximum at about 340 nm; (.) the quenchable component with a maximum at about 343 nm, characterized by an average value of K SV1 ¼ 5.02 M )1 and a fraction f 1 ¼ 0.81; and ( ) the unquenchable com- ponent with a maximum at about 337 nm, characterized by an aver- age value of K SV2 ¼ 0.00 M )1 and a fraction f 2 ¼ 0.19. M. Tworzydło et al. CRP conformational changes induced by DNA and cAMP FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS 1109 residue microenvironments [20]. To resolve the fluores- cence emission spectra into components in a protein containing multiple tryptophan residues, advanced techniques for analyzing fluorescence decay emission may be used [20]. Under steady-state conditions, the quenching processes may be analyzed by the external quenchers by using the FQRS method [21,22]. Quench- ing experiments are especially useful in studying the changes in the conformation of proteins that may be induced by ligand binding. If the studied protein pos- sesses several tryptophan residues, then the interpret- ation of a change in the quenchability is more difficult. However, site-directed mutagenesis may be used to obtain a single tryptophan-containing mutant protein, which will allow for a more straightforward interpret- ation of fluorescence quenching data. In this study, we used site-directed mutagenesis to obtain the CRPW85A mutant and used the FQRS method to observe conformational changes in the pro- tein upon binding of cAMP and fragments of DNA possessing specific sequences. Each CRP wild-type (CRPwt) subunit contains two tryptophan residues at positions 13 and 85, both located in the N-terminal domain of the protein [11,23]. Our previous fluores- cence quenching investigations [24] of CRPwt have shown that in apo–CRP, % 80% of the tryptophan fluorescence emission can be attributed to Trp13 and 20% of the fluorescence emission originates from Trp85. Our recently presented data concerning CRP mutants containing a single Trp13 or Trp85 residue indicate that binding of cAMP to anti sites in the CRP–(cAMP) 2 complex leads to changes in the Trp13 microenvironment, whereas its binding to syn sites in the CRP–(cAMP) 4 complex alters the surroundings of Trp85 [7]. The results presented in this report provide further evidence that binding of cAMP to the anti site of CRP induces local structural changes in the vicinity of Table 2. Fluorescence lifetimes and bimolecular quenching constants values for CRPW85A, CRPW85A–(cAMP) 2 and CRPW85A–DNA com- plexes. Experiments were performed at 20 °C in Tris buffer, pH 7.9. In the experiments with CRPW85A complexed to cAMP and DNA, the concentration of cAMP was 100 l M. Excitation was at 295 nm and emission through a cut-off filter. The error did not exceed 5%. Species s 1 (ns) f 1 s 2 (ns) s m (ns) Iodide quenching Acrylamide quenching k q1 (M )1 Æs )1 ) x10 )1 k q2 (M )1 Æs )1 ) x10 )1 k q1 (M )1 Æs )1 ) x10 )1 CRPW85A 3.09 0.69 0.58 2.31 4.16 0.73 2.49 CRPW85A–(cAMP) 2 2.99 0.65 0.55 2.14 4.67 1.23 3.09 CRPW85A–ICAP 2.54 0.59 0.29 1.62 4.60 – 3.92 CRPW85A–lac 4.23 0.57 0.62 2.68 2.07 – 2.29 CRPW85A–gal 3.93 0.51 0.62 2.31 2.17 – 3.39 Fig. 9. Kinetics of binding between IAEDANS-labeled CRP and fluo- rescein-labeled DNA, as measured by stopped-flow fluorymetry of the Fo ¨ rster resonance energy transfer (FRET). Measurements were performed at 20 °C, in buffer B, pH 8.0, with a DNA concentration of 0.2 l M:(d) lacF;(,) galF;(r) ICAPF. Excitation was at 340 nm and emission > 500 nm. Table 3. Kinetic and thermodynamic parameters describing the binding of lacF, galF and ICAPF to the wild-type cyclic AMP recep- tor protein (CRPwt). The values are derived from experiments con- ducted at 20 °C, in 50 m M Tris ⁄ HCl buffer, containing 100 mM KCl, 1m M EDTA, pH 8.0, in the presence of 200 lM cAMP. Kinetic and thermodynamic parameters are defined as detailed in the Experi- mental procedures. The error is the SD of fitted parameters. Complex k off (s )1 ) k on (s )1 ÆM )1 ) · 10 6 K a (M )1 ) · 10 5 CRPwt–ICAPF 5.8 ± 0.6 3.4 ± 0.2 5.9 ± 0.9 CRPwt–lacF 8.5 ± 0.9 1.1 ± 0.2 1.2 ± 0.3 CRPwt–galF 5.1 ± 0.9 2.4 ± 0.2 4.7 ± 0.9 CRP conformational changes induced by DNA and cAMP M. Tworzydło et al. 1110 FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS Trp13. Our fluorescence quenching measurements of apo–CRPW85A with iodide demonstrate that the steady-state fluorescence spectra of Trp13 can be resolved into two components by using the FQRS method. This result clearly shows that CRP exists in two distinct conformational states, each of which is characterized by a different microenvironment of Trp13. One of these states is characterized by its own fluorescence emission spectra with a maximum at 350 nm and the second state is characterized by a maximum emission spectrum at 338 nm. These two forms of the protein account for 55% and 45% of the total fluorescence emission, respectively. In contrast to the Trp13 residue, the tryptophan located at position 85 is characterized by one distinct fluorescence spec- trum (data not shown). The conformational state of apo–CRP, which possesses a maximum of the fluores- cence emission spectrum at 350 nm, can be character- ized by a Trp13 Stern–Volmer quenching constant, K SV ¼ 9.6 m )1 . If the average lifetime of Trp13 is assumed to be 2.3 ns, then the bimolecular rate- quenching constant, k q , can be calculated as 4.16 · 10 9 m )1 Æs )1 . This value is typical of the trypto- phan residues in proteins exposed to a solvent [25]. The second conformational state of CRP can be char- acterized by a relatively bluer emission with the maxi- mum at 338 nm. In this conformational state of CRP, the Trp13 residue is much less accessible to the iodide quencher, as can be judged by a bimolecular rate quenching constant, k q ¼ 0.73 · 10 9 m )1 Æs )1 . These two conformational states of CRP are not distinguish- able by acrylamide (another quencher used in this study). The acrylamide bimolecular rate quenching constant, k q , equaling 2.49 · 10 9 m )1 Æs )1 , is almost half that of the iodide rate-quenching constant. It has been well documented that nonionic acrylamide can penet- rate into the matrix of globular protein by diffusion, which is facilitated by small-amplitude fluctuations in the protein structure [25,26]. The process of quenching the fluorescence of Trp residues in protein by acryl- amide is more effective than by using the iodide ion [25,26]. Resolving the component spectra of the Trp13 resi- due of CRPW85A by using the FQRS method and fluorescence lifetime measurements enabled us to com- pare the fractional contributions of the fluorescence of the red and blue components from the solute quench- ing experiments by using the fractional contributions of the short and long lifetimes of the Trp13 residue obtained by lifetime measurements. A comparison of the fractional contribution values presented in Tables 1 and 2 shows a significant discrepancy, which suggests that the two Trp13 residues present in the CRPW85A homodimer do not fluoresce independently and that there is an energy transfer between them. A similar observation has been drawn from the resolved fluores- cence lifetime and solute quenching measurements per- formed for several two-tryptophan-containing proteins [27]. It may also be supposed that the fluorescence decay of the Trp13 residue is more complex than that described by a double-exponential decay, but we have had little success in trying to resolve the fluorescence to more components on our apparatus. As a result, when we calculated the bimolecular rate quenching constants, k q , we obtained values of the average Trp13 lifetime instead of the values of lifetimes of the resolved components. Binding of cAMP to anticAMP-binding sites leads to significant changes in the fluorescing properties of Trp13 of CRP–(cAMP) 2 , including changes in the maximum fluorescence emission of the component more quenchanable by iodide, as well as the increase in bimolecular rate-quenching constants, k q , for iodide and acrylamide (Tables 1 and 2). These results provide further evidence for changes in the protein dynamics induced by cAMP binding to the anti sites of CRP in the CRP–(cAMP) 2 complex, in the surroundings of Trp13. As the distance between the Trp13 residue and the anticAMP molecule, both located in the N-terminal domain in the CRP–(cAMP) 2 complex, is % 25.5 A ˚ [6], the observed changes in Trp13 fluorescence quenching by iodide and acrylamide result from the transduction of the conformational changes in the protein moiety and increase the dynamic motion around the Trp13 residue. This observation is in congruence with our previous time-resolved anisotropy fluorescence meas- urements of CRP, which show that cAMP binding to the protein leads to an increase in the structural dynamic motion around Trp13 [7]. As the Trp13 resi- due is close to the activation region of CRP, AR2, which is responsible for the interaction of the protein with the a subunit of RNA polymerase, it may be argued that the changes in the CRP dynamics in this molecule region can play an important role in signal transmission in the protein. Similarly, it has been shown that the Trp13 residue in CRP is directly engaged in the formation of the CRP complex with another gene-regulatory protein, such as CytR, in the CRP–CytR–DNA complex [28]. It is well established that the CRP allosteric activa- tion involves conformational changes that are trans- mitted from the N-terminal domain to the C-terminal domain of the protein and, in consequence, enable CRP to recognize the specific DNA sequences [2,4,11]. The results presented in this work provide evi- dence for conformational signal transduction in the M. Tworzydło et al. CRP conformational changes induced by DNA and cAMP FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS 1111 CRP–(cAMP) 2 complex after binding specific DNA, which occurs from the C-terminal domain to the N-terminal domain of the protein. We have shown this by using the Trp13-containing mutant of CRP as well as the iodide and acrylamide fluorescence quenching method in order to follow the influence of DNA bind- ing on the conformational changes in its microenviron- ment. We have used various DNA sequences: lac, gal and ICAP. The synthetic artificial ICAP DNA posses- ses a symmetrical sequence, which binds with high affinity to the CRP HTH motifs, and the lac and gal DNA sequences represent the CRP-binding sites from class I and class II CRP-dependent promoters, respect- ively [1]. Our iodide fluorescence-quenching measure- ments of DNA–CRP complexes show that CRP still exists in two different conformational states, but they significantly differ in Trp13 microenvironments which determine the Trp13 fluorescing properties. These dif- ferences do not result from an increase in the ionic strength of the solution upon titration of the sample by KI, because the titration performed with KCl up to a concentration similar to that of KI did not cause any change in fluorescence of the complexes (data not shown). The best fits for all the tested DNA sequences, as judged by reduced v 2 values as well as residual dis- tribution, have been obtained for two CRP states: one with an iodide-quenchable and the second with an iodide-unquenchable Trp13 residue. Binding DNA sequences to CRP causes only a small change in the maximum of the two resolved fluorescence emission spectra, but shows that the iodide-quenchable compo- nents account for % 75% of the total emission of Trp13, in comparison to % 55% in the CRP–(cAMP) 2 complex (Table 1). As the binding of the tested DNA sequences also leads to changes in the average fluores- cence lifetime of Trp13, it may be expected that the observed changes result from both the static and dynamic processes that occur in the microenviron- ments of this residue. Thr10, Asp109 and His17, which are located within a distance up to 5 A ˚ [29] are the most probable candidates as quenching residues of CRP, in the vicinity of Trp13. The accessibility for iodide as well as acrylamide, expressed by k q values (Table 2), differs for the three studied DNA sequences and clearly shows that binding of the particular DNA to CRP causes different local changes in Trp13 residue exposition. As this residue is located close to the acti- vation region, AR2, which is responsible for the inter- action with the RNA polymerase, it is tempting to suggest that the binding of CRP to the DNA promoter in solution involves a further conformational signal transduction from the C-terminal domain to the N-ter- minal domain of CRP, and the magnitude of this conformational transduction solely depends on the promoter DNA sequence responsible for this inter- action. This suggestion is in agreement with small angle neutron scattering measurements of the CRP– DNA complex, which indicate that this structural change in the N-terminal domain of the protein occurs upon binding of DNA to the C-terminal domain of CRP [30]. Our fluorescence studies of CRP–DNA interactions presented here also agree with the results of Baichoo & Heyduk [31], which were obtained by protein footprinting techniques. These authors, using chemical proteases of different charge, size and hydro- phobicity, suggested that the binding of DNA in solu- tion induces conformational changes in the N-terminal domain of CRP, close to the activating region, AR2. Our fast-kinetics study presented here has also shown that the DNA–CRP interactions depend on the sequence of the 26 bp DNA fragments. The bimole- cular rate constant values of 3.4 · 10 6 m )1 Æs )1 , 1.1 · 10 6 m )1 Æs )1 and 2.4 · 10 6 m )1 Æs )1 , determined for ICAP, lac and gal, respectively, are very similar to the values of rate constants calculated for the interaction of DNA with other proteins [32–34]. However, the monomolecular dissociation rate constants determined for the CRP–ICAP, CRP–lac and CRP–gal complexes, of 5.8 s )1 , 8.5 s )1 and 5.1 s )1 , respectively, are signifi- cantly higher than the range between 10 )3 and 10 )2 s )1 that has been found for other proteins which interact with DNA [32–34]. The observed differences in the dis- sociation rate constants may result from the fast association of CRP with DNA, which leads to forma- tion of the low-affinity CRP–DNA complex. This is followed by the slow process of conformational chan- ges in the C-terminal domains of CRP, which permit formation of the high-affinity complex. As the kinetics of CRP–DNA interactions have been detected by determining the resonance energy transfer between fluorescently labeled CRP and DNA, we have been able to observe only the first step of the association process without detecting any possible consecutive reactions. However, we have observed the fluorescence intensity changes of CRP–AEDANS upon the binding of DNA sequences, which result from the conforma- tional changes in the C-terminal domain of the pro- tein. The values of CRP–DNA association equilibrium constants, K a , calculated from the rate constants pre- sented in Table 3, are equal to 5.9 · 10 5 m )1 , 1.2 · 10 5 m )1 and 4.7 · 10 5 m )1 for ICAP, lac and gal, respectively. These values are slightly lower than the association constants of 4.0 · 10 5 m )1 and 11.1 · 10 5 m )1 that were determined by isothermal titration calorimetry for lac and gal, respectively [35]. The 26 bp long DNA sequences – lac, gal and ICAP – have CRP conformational changes induced by DNA and cAMP M. 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