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• W2007989871 abstract "The cAMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. After allosteric transitions resulting from the binding of cAMP, CRP specifically binds to DNA and activates transcription. We have used stopped-flow fluorometry measurements of CRP mutants bearing amino acid substitutions T127I, S128A, and T127I/S128A to study the kinetics of conformational changes in the protein induced by cAMP binding. Amino acid substitutions at positions 127 and 128 were chosen because these residues play a crucial role in interdomain and intersubunit communication during allosteric transition. Using N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonic acid-labeled Cys178, localized in the protein helix-turn helix motif, we observed conformational changes in the helix-turn helix, localized in the C-terminal domain, upon binding of cAMP to high affinity sites (CRP·cAMP2) in the N-terminal domain of CRP. The rate constants for the forward and backward conformational changes depend on the amino acid substitution: kc = 3.62 s–1 and k –c = 3.13s–1 for CRP T127I and k c = 0.42 s–1 and k – c = 0.78 s–1 for CRP S128A. These values can be compared with kc = 9.7 s–1 and k – c = 0.31 s–1 for wild-type CRP. The observed conformational changes can be described by the sequential model of allostery, with the amino acid substitutions influencing the allosteric changes. In the case of the double mutant, the observed rate constant of cAMP binding supports the suggestion that this unligated mutant possesses the structure that is close to the allosteric conformation necessary for promoter binding. The results of intrinsic fluorescence measurements suggest that the formation of the CRP·cAMP4 complex results from displacement of equilibrium between the two forms of the CRP·cAMP2 complex in the mutants studied, similar to wild-type CRP. The observed conformational changes occur according to a concerted model of allostery, and isomerization equilibrium between the two CRP states depends on the amino acid substitution. The data presented in this study indicate that Ser128 and Thr127 in CRP play an important role in the kinetics of intramolecular transitions triggered by cAMP. The cAMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. After allosteric transitions resulting from the binding of cAMP, CRP specifically binds to DNA and activates transcription. We have used stopped-flow fluorometry measurements of CRP mutants bearing amino acid substitutions T127I, S128A, and T127I/S128A to study the kinetics of conformational changes in the protein induced by cAMP binding. Amino acid substitutions at positions 127 and 128 were chosen because these residues play a crucial role in interdomain and intersubunit communication during allosteric transition. Using N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonic acid-labeled Cys178, localized in the protein helix-turn helix motif, we observed conformational changes in the helix-turn helix, localized in the C-terminal domain, upon binding of cAMP to high affinity sites (CRP·cAMP2) in the N-terminal domain of CRP. The rate constants for the forward and backward conformational changes depend on the amino acid substitution: kc = 3.62 s–1 and k –c = 3.13s–1 for CRP T127I and k c = 0.42 s–1 and k – c = 0.78 s–1 for CRP S128A. These values can be compared with kc = 9.7 s–1 and k – c = 0.31 s–1 for wild-type CRP. The observed conformational changes can be described by the sequential model of allostery, with the amino acid substitutions influencing the allosteric changes. In the case of the double mutant, the observed rate constant of cAMP binding supports the suggestion that this unligated mutant possesses the structure that is close to the allosteric conformation necessary for promoter binding. The results of intrinsic fluorescence measurements suggest that the formation of the CRP·cAMP4 complex results from displacement of equilibrium between the two forms of the CRP·cAMP2 complex in the mutants studied, similar to wild-type CRP. The observed conformational changes occur according to a concerted model of allostery, and isomerization equilibrium between the two CRP states depends on the amino acid substitution. The data presented in this study indicate that Ser128 and Thr127 in CRP play an important role in the kinetics of intramolecular transitions triggered by cAMP. The cAMP receptor protein (CRP) 1The abbreviations used are: CRP, cAMP receptor protein; HTH, helix-turn-helix; KNF, Koshland-Némethy-Filmer (sequential model of allostery); MWC, Monod-Wyman-Changeux (concerted model of allostery); I-EDANS, N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonic acid; EDANS-CRP, cAMP receptor protein covalently labeled with I-EDANS at Cys178. regulates the expression of >100 genes in Escherichia coli. CRP is a dimeric protein composed of two identical subunits of 209 amino acid residues with a molecular mass of 22.6 kDa (1Busby S. Ebright R. J. Mol. Biol. 1999; 293: 199-213Crossref PubMed Scopus (644) Google Scholar, 2Harman J.G. Biochim. Biophys. Acta. 2001; 1574: 1-17Crossref Scopus (212) Google Scholar). Each subunit is folded into two distinct domains (3Anderson W.B. Schneider A.B. Emmer M. Perlman R.L. Pastan I. J. Biol. Chem. 1971; 246: 5929-5937Abstract Full Text PDF Google Scholar). The larger N-terminal domain contains a binding site for cAMP in the anti-conformation, and the smaller C-terminal domain contains a helix-turn-helix (HTH) motif responsible for DNA recognition and binding. The two domains are connected with a short hinge region composed of residues 135–138. Crystal structure studies have shown that the protein additionally possesses a second binding site (located between the hinge and the turn of HTH) that binds cAMP in the syn-conformation (4Passner J.M. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2843-2847Crossref PubMed Scopus (161) Google Scholar). Indeed, a recent microcalorimetric isothermal titration calorimetry study has shown that, in solution, CRP sequentially binds two molecules of cAMP with negative cooperativity and high affinity; and additionally, at least one additional molecule binds to a low affinity binding site (5Lin S.H. Lee J.C. Biochemistry. 2002; 41: 11857-11867Crossref PubMed Scopus (55) Google Scholar). It is believed that, at ∼100 μm cAMP, CRP is able to recognize and bind specific DNA sequences and to stimulate transcription (6Taniguchi T. O'Neill M. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5090-5094Crossref PubMed Scopus (104) Google Scholar), and this active form of the protein is represented by the CRP·cAMP2 complex with two anti-sites occupied (7Mukhopadhyay J. Sur R. Parrack P. FEBS Lett. 1999; 453: 215-218Crossref PubMed Scopus (29) Google Scholar, 8Małecki J. Polit A. Wasylewski Z. J. Biol. Chem. 2000; 275: 8480-8486Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). However, in the presence of millimolar concentrations of cAMP, at which the CRP·cAMP4 complex predominates, there is a loss of affinity for DNA and sequence specificity for its recognition and transcription regulation (7Mukhopadhyay J. Sur R. Parrack P. FEBS Lett. 1999; 453: 215-218Crossref PubMed Scopus (29) Google Scholar). Therefore, the occupancy of cAMP-binding anti- or syn-sites of CRP may have different effects on the regulatory mechanism of CRP associated with interactions between the protein subunits and domains. The idea of four cAMP-binding sites, viz. two in its anti-conformation operating at low concentrations of the ligand and two additional binding sites active at high cAMP concentrations, has also been used to describe the results of our fast kinetic studies (8Małecki J. Polit A. Wasylewski Z. J. Biol. Chem. 2000; 275: 8480-8486Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) as well as time-resolved anisotropy (9Błaszczyk U. Polit A. Guz A. Wasylewski Z. J. Protein Chem. 2002; 20: 601-610Crossref Scopus (12) Google Scholar) and fluorescence energy transfer measurements (10Polit A. Błaszczyk U. Wasylewski Z. Eur. J. Biochem. 2003; 270: 1413-1423Crossref PubMed Scopus (15) Google Scholar). A variety of biochemical and biophysical studies (2Harman J.G. Biochim. Biophys. Acta. 2001; 1574: 1-17Crossref Scopus (212) Google Scholar) have demonstrated that binding of cAMP allosterically induces CRP to assume a conformation that binds to DNA and that interacts with RNA polymerase (1Busby S. Ebright R. J. Mol. Biol. 1999; 293: 199-213Crossref PubMed Scopus (644) Google Scholar). However, the details of the mechanism, which mediates the allosteric activation of CRP, remain obscure because the crystal structure of unligated CRP has not yet been elucidated. It is believed that the binding of cAMP involves subunit realignment and a reorientation of the hinge between the protein domains (11Won H.-S. Yamazaki T. Lee T.-W. Yoon M.-K. Park S.-H. Kyogoku Y. Lee B.-J. Biochemistry. 2000; 39: 13953-13962Crossref PubMed Scopus (59) Google Scholar). Our recent fluorescence energy transfer studies support this idea and show that the binding of anti-cAMP in the CRP·cAMP2 complex results in the movement of the C-terminal domain of CRP by ∼8 Å toward the N-terminal domain (10Polit A. Błaszczyk U. Wasylewski Z. Eur. J. Biochem. 2003; 270: 1413-1423Crossref PubMed Scopus (15) Google Scholar). The binding of cAMP in the CRP·cAMP4 complex effects only a very small increase in fluorescence energy transfer efficiency between fluorescently labeled Cys178 and Trp85. Biochemical and biophysical studies focused on the identification of structural changes accompanying binding of cAMP to CRP have been mostly performed under equilibrium conditions, and much less attention has been paid to the description of the kinetic mechanism of the ligand interaction with CRP. Our previous stopped-flow studies investigating the kinetics of conformational changes in CRP induced by cAMP binding had shown that, at micromolar cAMP concentrations, allosteric changes take place according to a sequential (Koshland-Némethy-Filmer (KNF)) model, whereas conformational changes observed at millimolar concentrations can be described by a concerted (Monod-Wyman-Changeux (MWC)) model (8Małecki J. Polit A. Wasylewski Z. J. Biol. Chem. 2000; 275: 8480-8486Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The conformational change observed upon binding of cAMP to anti-sites, described by the KNF model, clearly indicates long-range structural communication between the N- and C-terminal domains of CRP remaining under kinetic control (8Małecki J. Polit A. Wasylewski Z. J. Biol. Chem. 2000; 275: 8480-8486Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). On the other hand, binding of cAMP to syn-sites of CRP, described by the MWC model, results from displacement of equilibrium between the two structural forms of CRP·cAMP2, where only one form is able to form a CRP·cAMP4 complex. Because the four cAMP-binding sites of CRP possess different cooperative affinity for the ligand (8Małecki J. Polit A. Wasylewski Z. J. Biol. Chem. 2000; 275: 8480-8486Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), it can be expected that the kinetically controlled domain-domain and subunit-subunit communication may play a crucial role in the molecular regulatory mechanism of CRP action. In this study, we report a further kinetic description of cAMP-induced conformational changes in CRP based on the use of different mutants (T127I, S128A, and T127I/S128A). It is well known (12Cheng X. Kovac L. Lee J.C. Biochemistry. 1995; 34: 10816-10826Crossref PubMed Scopus (38) Google Scholar, 13Leu S.W. Baker C.H. Lee E.J. Harman J.G. Biochemistry. 1999; 38: 6222-6230Crossref PubMed Scopus (31) Google Scholar, 14Chu S. Tordova M. Gilliland G.L. Gorshkova I. Shi Y. Wang S. Schwarz F.P. J. Biol. Chem. 2001; 276: 11230-11236Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) that all of these mutants exhibit altered allosteric activation of CRP both in vitro and in vivo (15Lee E.J. Glasgow J. Leu S.F. Belduz A.O. Harman J.G. Nucleic Acids Res. 1994; 22: 2894-2901Crossref PubMed Scopus (46) Google Scholar). Because Thr127 and Ser128 are directly involved in cAMP binding as well as in intersubunit and interdomain interactions in CRP·cAMP complexes (4Passner J.M. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2843-2847Crossref PubMed Scopus (161) Google Scholar, 16Weber I.T. Steitz T.A. J. Mol. Biol. 1987; 198: 311-326Crossref PubMed Scopus (412) Google Scholar), fast kinetic measurements can provide further insight into the allosteric activation of CRP. Materials—N-Iodoacetylaminoethyl-5-naphthylamine-1-sulfonic acid (I-EDANS) was purchased from Molecular Probes, Inc. EDTA, dithiothreitol, phenylmethylsulfonyl fluoride, Tris, and cAMP were obtained from Sigma. Fractogel EMD SO3- 650M was from Merck, and Q-Sepharose Fast Flow, Sephacryl S-200HR, and Sephadex G-25 were from Amersham Biosciences. The nutrients for bacterial growth were from Invitrogen. All other chemicals were analytical grade products from ICN. All measurements were performed in buffers prepared in water purified with the Millipore system. Protein Purification—CRP S128A was isolated from E. coli strain CA8445 transformed with plasmid pPLc28, which encodes a mutant crp gene. The mutation at position 127 (threonine replaced by isoleucine) as well as the double mutation T127I/S128A were performed using the overlap extension method with Pwo polymerase. Plasmid pHA7, which encodes mutant crp genes, was introduced into E. coli M182Δcrp. Cells were grown on LB medium at 37 °C (M182Δcrp) or 28 °C (CA8445) in a Biostat B fermentor (B. Braun Biotech International). CRP S128A expression was induced in the host bacteria by shifting the temperature from 28 to 42 °C. All proteins were purified at 4 °C essentially as described previously (8Małecki J. Polit A. Wasylewski Z. J. Biol. Chem. 2000; 275: 8480-8486Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), with one modification. After ion-exchange chromatography on Q-Sepharose, the protein was additionally purified by gel filtration on Sephacryl S-200HR. After the purification procedure, the protein was highly pure (>97%) as judged by SDS-PAGE and Coomassie Brilliant Blue staining. Measurements were performed in 50 mm Tris-HCl (pH 8.0) containing 100 mm KCl and 1 mm EDTA (buffer A) or 50 mm Tris-HCl (pH 7.8) supplemented with 100 mm KCl and 1 mm EDTA (buffer B). The following absorption coefficients were used: 14,650 m–1 cm–1 at 259 nm for cAMP (17Merck & Co., Inc.The Merck Index. 9th ed. Merck & Co., Inc., Rahway, NJ1976Google Scholar), 20,400 m–1 cm–1 at 278 nm for CRP monomer (18Takahashi M. Blazy B. Baudras A. Biochemistry. 1980; 19: 5124-5130Crossref PubMed Scopus (99) Google Scholar), and 6000 m–1 at 340 nm for I-EDANS (19Hudson E.N. Weber G. Biochemistry. 1973; 12: 4154-4161Crossref PubMed Scopus (392) Google Scholar). Fluorescence Labeling of CRP—Details of the preparation of I-EDANS-labeled CRP T127I, CRP S128A, and CRP T127I/S128A were described previously (10Polit A. Błaszczyk U. Wasylewski Z. Eur. J. Biochem. 2003; 270: 1413-1423Crossref PubMed Scopus (15) Google Scholar). The labeled proteins were purified on a Sephadex G-25 column equilibrated with buffer A. Fractions displaying absorbance at both 280 and 340 nm were combined and dialyzed extensively against buffer A. The stoichiometry of labeling was determined spectrophotometrically and was in the range of 1.0–1.8 mol of label/mol of CRP dimer. It has been shown previously that only Cys178 (C-terminal domain) can be chemically modified under conditions preserving its native structure (10Polit A. Błaszczyk U. Wasylewski Z. Eur. J. Biochem. 2003; 270: 1413-1423Crossref PubMed Scopus (15) Google Scholar, 20Eilen E. Krakow J.S. J. Mol. Biol. 1977; 114: 47-60Crossref PubMed Scopus (44) Google Scholar). Stopped-flow Fluorescence Measurements—The stopped-flow fluorescence experiments were performed using an SX-17 MV stopped-flow spectrophotometer (Applied Photophysics, Leatherhead, UK) in the two-syringe mode. The dead time of mixing was determined to be <2 ms. Conformation changes in the proteins induced by cAMP binding were monitored by the fluorescence intensity of I-EDANS covalently attached to Cys178 or by the fluorescence intensity of tryptophan residues (Trp13 and Trp85). CRP covalently labeled with I-EDANS at Cys178 (I-EDANS-CRP) was excited at 340 nm, and fluorescence emission was monitored at wavelengths >475 nm using a cutoff filter. The fluorescence intensity of tryptophan residues was observed at >320 nm using a cutoff filter after excitation at 295 nm. All measurements were performed at 20 ± 0.1 °C, and the reaction mixtures contained final concentrations of 1 μm I-EDANS-CRP and ∼2 μm CRP (for tryptophan experiments). Background measurements were also carried out at the beginning of each experiment. The sample was incubated in a stoppedflow syringe for 5 min to allow for thermal equilibration. Stopped-flow experiments were initiated by mixing equal volumes of protein (fixed concentration) and various concentrations of cAMP. Fourthousand data points were acquired in each stopped-flow experiment. Multiple kinetic runs were summed (10Polit A. Błaszczyk U. Wasylewski Z. Eur. J. Biochem. 2003; 270: 1413-1423Crossref PubMed Scopus (15) Google Scholar, 11Won H.-S. Yamazaki T. Lee T.-W. Yoon M.-K. Park S.-H. Kyogoku Y. Lee B.-J. Biochemistry. 2000; 39: 13953-13962Crossref PubMed Scopus (59) Google Scholar, 12Cheng X. Kovac L. Lee J.C. Biochemistry. 1995; 34: 10816-10826Crossref PubMed Scopus (38) Google Scholar, 13Leu S.W. Baker C.H. Lee E.J. Harman J.G. Biochemistry. 1999; 38: 6222-6230Crossref PubMed Scopus (31) Google Scholar, 14Chu S. Tordova M. Gilliland G.L. Gorshkova I. Shi Y. Wang S. Schwarz F.P. J. Biol. Chem. 2001; 276: 11230-11236Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) to obtain adequate signal-to-noise ratios. Such averaged kinetic traces were fit to a single exponential or to a sum of such terms (Equation 1), F(t)=Aexp(-kt)+C(Eq. 1) where F is the fluorescence intensity at time t; A and k are the amplitude and observed rate constant, respectively; and C is the fluorescence at infinite time. All kinetic traces were analyzed using software supplied by Applied Photophysics. The validity of the fit was evaluated by an inspection of residuals and normalized variation parameters. Binding of cAMP Monitored by I-EDANS Fluorescence—The kinetics of the binding of cAMP to the CRP T127I, CRP S128A, and CRP T127I/S128A mutants were measured as changes in the I-EDANS-CRP fluorescence using a stopped-flow method under pseudo first-order conditions. In the absence of cAMP, after excitation at 340 nm, the maximum fluorescence emission of I-EDANS was observed at ∼490 nm for all of the labeled CRP mutants. In all cases, after adding cAMP, an increase in I-EDANS fluorescence was observed with increasing ligand concentrations. The maximum increases in the fluorescence intensity of the I-EDANS moiety observed in the kinetic experiments were ∼16.5 and ∼7% for CRP T127I and CRP S128A, respectively. In addition, a blue shift occurred in the emission spectra, shifting the maximum to ∼478 and ∼480 nm and for CRP T127I and CRP S128A, respectively. The fluorescence intensity changes and the blue shift suggest that the conformational changes induced by cAMP in CRP T127I and CRP S128A are different. In the case of the CRP double mutant, the addition of the ligand did not cause significant changes in the fluorescence intensity of I-EDANS. The progress curves of the reactions of CRP T127I and CRP S128A with cAMP fit well with the single-exponential model. The addition of a second exponent did not significantly alter the goodness of the fit. The normalized variance value (obtained with the single- and double-exponential fits) indicates that the two-component analysis is not significantly better than the one-component analysis. Also, the distribution of the residuals was not improved upon the addition of the second exponent. Fig. 1 shows a typical kinetic course of the binding reaction between cAMP and CRP T127I. For the CRP T127I and CRP S128A mutants, the observed rate constants (k obs) increased with increasing ligand concentrations and approached a constant value at high millimolar concentrations of cAMP, as shown in Figs. 2 and 3. Thus, they are consistent with our previous kinetic studies using wild-type CRP (8Małecki J. Polit A. Wasylewski Z. J. Biol. Chem. 2000; 275: 8480-8486Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar).Fig. 2Dependence of observed rate constants (k obs) for cAMP-induced changes in the fluorescence intensity of I-EDANS-CRP T127I on the final cAMP concentration. The solid line is the best fit according to Equation 2, with the parameters of the fit summarized in Table I.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Observed rate constants (k obs) for the association of CRP S128A with cAMP measured by changes in the fluorescence intensity of the I-EDANS label. The solid line is the best fit according to Equation 2, with the parameters of the fit summarized in Table I.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As we described previously (8Małecki J. Polit A. Wasylewski Z. J. Biol. Chem. 2000; 275: 8480-8486Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), hyperbolic saturation of k obs versus the cAMP concentration would indicate a two-step binding process. After a very fast binding process, the doubleligated protein undergoes conformational changes, which were observed in our measurements. Thus, the increase in rate constants accompanying the binding of cAMP at micromolar concentrations to CRP S128A and CRP T127I was analyzed in terms of a sequential (KNF) model of allostery, represented by Scheme 1, are intrinsic dissociation constants for binding of the first and second cAMP molecules to high affinity (anti) sites, respectively; and CRP and CRP* represent the protein before and after the conformational change, respectively. KC is the equilibrium constant between these two forms, whereas kc and k – c are rate constants that describe the conformational change step. If cAMP binding occurs with a simple two-step mechanism, assuming independent binding, as shown in Scheme 1, then the observed rate constant (k obs) should increase with increasing cAMP concentrations according to Equation 2. kobs=k-c+kc[cAMP]2Kanti2+2Kanti[cAMP]+[cAMP]2(Eq. 2) In Equation 2, Kanti has been considered as a geometric average of K 1 and K 2. The resultant parameters of fitting the data to Equation 2 are shown in Table I.Table IKinetic and thermodynamic parameters describing cAMP binding to high affinity sites of CRP T127I and CRP S128AProteinkck-cKCKantis-1s-1μ mWild-type CRPaData are from Ref. 8.9.7 ± 0.10.31 ± 0.050.03227.5 ± 1.0CRP T127I3.62 ± 0.253.13 ± 0.260.8620.1 ± 2.5CRP S128A0.42 ± 0.120.78 ± 0.131.8636 ± 24a Data are from Ref. 8Małecki J. Polit A. Wasylewski Z. J. Biol. Chem. 2000; 275: 8480-8486Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar. Open table in a new tab Binding of cAMP Monitored by Fluorescence of Tryptophan Residues—The rate of cAMP binding to CRP T127I, CRP S128A, and CRP T127I/S128A was also measured by fluorescence intensity of tryptophan residues using a stopped-flow method. As we have shown previously (8Małecki J. Polit A. Wasylewski Z. J. Biol. Chem. 2000; 275: 8480-8486Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), binding of cAMP to wild-type CRP caused significant changes in the Trp intensity only at high ligand concentrations (>300 μm). Similar observations were made with the CRP S128A mutant. However, pronounced changes in the fluorescence intensity of the tryptophan residues of CRP T127I and CRP T127I/S128A were detected only at very high concentrations of cAMP, viz. 3 and 10 mm, respectively. In all cases, mixing cAMP with CRP T127I, CRP S128A, and CRP T127I/S128A resulted in a time-dependent increase in fluorescence emission by the tryptophan residues. The fluorescence intensity increased by ∼5, 9, and 3% for CRP T127I, CRP S128A, and CRP T127I/S128A, respectively. This suggests that the conformational changes induced by cAMP in CRP T127I, CRP S128A, and CRP T127I/S128A are different. Fig. 4 presents the progress curves for association of CRP T127I, CRP S128A, and CRP T127I/S128A with 10 mm cAMP. For all concentrations of cAMP, the kinetic traces could be fitted well using a single-exponential curve; double-exponential fitting did not improve the goodness of the fit. The rate constants (k obs) calculated from the kinetic traces were plotted against the concentration of the ligand. For all investigated CRP mutants, as the cAMP concentration increased, the observed rate constants decreased, reaching a plateau at very high ligand concentrations, as shown in Fig. 5. A good description of the CRP T127I, CRP S128A, and CRP T127I/S128A interactions with cAMP was obtained using a model that we described in our previous study, where we analyzed the binding of cAMP to wild-type CRP (8Małecki J. Polit A. Wasylewski Z. J. Biol. Chem. 2000; 275: 8480-8486Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). As we have reported, at millimolar cAMP concentrations, CRP undergoes a conformational change according to a concerted (MWC) model of allostery, which can be represented by Scheme 2, where P′ and P are the binding and non-binding forms of protein; K 0 is the equilibrium constant between these two states; k on and k off are rate constants that describe the isomerization step; and K 3 and K 4 are intrinsic dissociation constants for binding of the first and second cAMP molecules to low affinity (syn) sites, respectively. The association of cAMP with CRP is a very fast process, and P and P′ are two interconvertible forms of the CRP·cAMP2 complex. The changes in fluorescence intensity of Trp residues upon the addition of cAMP observed in our experiment must be associated with the slow isomerization of the CRP·cAMP2 complex after its formation. Moreover, as these changes occur at a millimolar concentration of cAMP, they must be associated with binding of the ligand to low affinity sites of the protein.Fig. 5Dependence of observed rate constants (k obs) for cAMP-induced changes in the Trp fluorescence intensity of CRP T127I, CRP S128A, and CRP T127I/S128A on the final cAMP concentration. □, CRP T127I; ▿, CRP S128A; ○, CRP T127I/S128A. The solid lines are the best fit according to Equation 3, with the parameters of the fit summarized in Table II.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Scheme 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) The observed rate constants (k obs) derived from kinetic experiments performed at millimolar cAMP concentrations can be fitted to Equation 3 to yield the kinetic and thermodynamic parameters k on, k off, and Ksyn. kobs=kon+koffKsyn2Ksyn2+2Ksyn[cAMP]+[cAMP]2(Eq. 3) In Equation 3, Ksyn has been considered as a geometric average of K 3 and K 4. The kinetic and thermodynamic parameters obtained from fitting the data to Equation 3 are reported in Table II.Table IIKinetic and thermodynamic parameters describing cAMP binding to low affinity sites of CRP T127I, CRP S128A, and CRP T127I/S128AProteinkonkoffK0Ksyns-1s-1mmWild-type CRPaData are from Ref. 8.28 ± 1.575.5 ± 3.02.72.0 ± 0.2CRP T127I1.4 ± 2.070.2 ± 10.1508.5 ± 2.3CRP S128A0.6 ± 1.947.5 ± 3.7792.7 ± 0.7CRP T127I/S128A8.4 ± 2.550.7 ± 34.469.1 ± 7.7a Data are from Ref. 8Małecki J. Polit A. Wasylewski Z. J. Biol. Chem. 2000; 275: 8480-8486Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar. Open table in a new tab The conformational changes induced by cAMP binding to high affinity anti-sites result in a switch of CRP from a low affinity nonspecific DNA-binding protein to a state of the protein that binds DNA with high affinity and sequence specificity. It has been accepted that this phenomenon is associated with CRP subunit-subunit realignment and hinge reorientation (11Won H.-S. Yamazaki T. Lee T.-W. Yoon M.-K. Park S.-H. Kyogoku Y. Lee B.-J. Biochemistry. 2000; 39: 13953-13962Crossref PubMed Scopus (59) Google Scholar), and our recent experiments have shown that this change is accompanied by an ∼8-Å decrease in the distance between the two domains of the protein (10Polit A. Błaszczyk U. Wasylewski Z. Eur. J. Biochem. 2003; 270: 1413-1423Crossref PubMed Scopus (15) Google Scholar). Our previous kinetic studies have demonstrated that the formation of the CRP·cAMP2 complex, after saturation of high affinity sites, is followed by a conformational change that occurs according to the sequential model of allostery (8Małecki J. Polit A. Wasylewski Z. J. Biol. Che" @default.
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• W2007989871 date "2003-10-01" @default.
• W2007989871 modified "2023-10-18" @default.
• W2007989871 title "Kinetic Studies of cAMP-induced Allosteric Changes in Mutants T127I, S128A, and T127I/S128A of the cAMP Receptor Protein from Escherichia coli" @default.
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• W2007989871 doi "https://doi.org/10.1074/jbc.m306398200" @default.
• W2007989871 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12939272" @default.
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