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- W2019898190 abstract "Cyclic AMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The ability of CRP to bind specific DNA sequences and stimulate transcription is achieved as result of binding of an allosteric ligand: cAMP. Stopped-flow fluorimetry was employed to study the kinetics of the conformational changes in CRP induced by cAMP binding to high and low affinity receptor sites. Results of experiments using CRP labeled at Cys-178 with 1,5-I-AENS indicate change in conformation of the helix-turn-helix, occurring after the formation of CRP-cAMP2 complex, i.e. after saturation of the high affinity sites. The observed conformational change occurs according to sequential model of allostery and is described by rate constants: kc = 9.7 ± 0.1 s−1 and k -c = 0.31 ± 0.05 s−1, for the forward and backward reaction, respectively. Results of experiments monitored using CRP intrinsic fluorescence suggest that conformational change precedes the formation of CRP-cAMP4 complex and results from displacement of equilibrium between two forms of CRP-cAMP2, caused by binding of cAMP to low affinity sites of one of these forms only. The observed conformational change occurs according to concerted model of allostery and is described by rate constants:k on = 28 ± 1.5 s−1 andk off = 75.5 ± 3 s−1. Results of experiments using single-tryptophan-containing CRP mutants indicate that Trp-85 is mainly responsible for the observed total change in intrinsic fluorescence of wild-type CRP. Cyclic AMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The ability of CRP to bind specific DNA sequences and stimulate transcription is achieved as result of binding of an allosteric ligand: cAMP. Stopped-flow fluorimetry was employed to study the kinetics of the conformational changes in CRP induced by cAMP binding to high and low affinity receptor sites. Results of experiments using CRP labeled at Cys-178 with 1,5-I-AENS indicate change in conformation of the helix-turn-helix, occurring after the formation of CRP-cAMP2 complex, i.e. after saturation of the high affinity sites. The observed conformational change occurs according to sequential model of allostery and is described by rate constants: kc = 9.7 ± 0.1 s−1 and k -c = 0.31 ± 0.05 s−1, for the forward and backward reaction, respectively. Results of experiments monitored using CRP intrinsic fluorescence suggest that conformational change precedes the formation of CRP-cAMP4 complex and results from displacement of equilibrium between two forms of CRP-cAMP2, caused by binding of cAMP to low affinity sites of one of these forms only. The observed conformational change occurs according to concerted model of allostery and is described by rate constants:k on = 28 ± 1.5 s−1 andk off = 75.5 ± 3 s−1. Results of experiments using single-tryptophan-containing CRP mutants indicate that Trp-85 is mainly responsible for the observed total change in intrinsic fluorescence of wild-type CRP. cyclic AMP receptor protein 5-I-AENS,N-iodoacetylaminoethyl-1-naphthylamine-5-sulfonate CRP covalently labeled at Cys-178 residues with 1,5-I-AENS dithiothreitol helix-turn-helix Koshland-Némethy-Filmer sequential model of allostery Monod-Wyman-Changeux concerted model of allostery wild type CRP1 regulates the expression of over 100 genes, located in several Escherichia coli operons, most of them being involved in the cell's response to glucose starvation conditions (1.Kolb A. Busby S. Buc H. Garges S. Adhya S. Annu. Rev. Biochem. 1993; 62: 749-795Crossref PubMed Google Scholar). The protein is a homodimer, composed of 209 amino acid residues per monomer, and each subunit is folded in two domains (2.Weber I.T. Steitz T.A. J. Mol. Biol. 1987; 198: 311-326Crossref PubMed Scopus (409) Google Scholar). The larger N-terminal domain contains a binding site for cAMP in anti conformation and is responsible for the dimer stability. The smaller C-terminal domain contains the HTH motif, responsible for DNA recognition and binding. The two domains are connected with a short hinge region (residues 135–138). A recent crystal structure of CRP-DNA complex showed, that each protein subunit binds two cAMP molecules (3.Passner J.M. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2843-2847Crossref PubMed Scopus (158) Google Scholar). In addition to previously known binding site, located in the N-terminal domain, there is a new site located between the hinge and the turn of HTH, which binds cAMP in the syn conformation. Several biochemical and biophysical properties of CRP exhibit a bimodal dependence on cAMP concentration (4.Heyduk T. Lee J.C. Biochemistry. 1989; 28: 6914-6924Crossref PubMed Scopus (116) Google Scholar). This has been interpreted as evidence for existence of three conformational states of the protein, namely: unliganded CRP, CRP-cAMP1 with one cAMP bound to ananti site, and CRP-cAMP2 with bothanti sites filled. Basing on their most recent crystal structure, Passner and Steitz (3.Passner J.M. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2843-2847Crossref PubMed Scopus (158) Google Scholar) reinterpreted those results in terms of three conformational states represented by CRP, CRP-cAMP2 with cAMP bound to two anti sites, and CRP-cAMP4 with both two anti and twosyn sites filled. In presence of ∼100 μm [cAMP], CRP becomes “activated” and is able to recognize and bind specific DNA sequences and stimulate transcription (5.Taniguchi T. O'Neill M. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5090-5094Crossref PubMed Scopus (104) Google Scholar). Crystallographic (2.Weber I.T. Steitz T.A. J. Mol. Biol. 1987; 198: 311-326Crossref PubMed Scopus (409) Google Scholar) and equilibrium dialysis studies (6.Takahashi M. Blazy B. Baudras A. Biochemistry. 1980; 19: 5124-5130Crossref PubMed Scopus (98) Google Scholar) indicate that the “active” form of the protein is represented by CRP-cAMP2 complex, with twoanti sites filled. However, in the presence of millimolar concentrations of cAMP, where CRP-cAMP4 complex predominates, there is a loss in affinity and sequence specificity in DNA binding and consequently loss in transcription stimulation (7.Mukhopadhyay J. Sur R. Parrack P. FEBS Lett. 1999; 453: 215-218Crossref PubMed Scopus (29) Google Scholar). This strongly suggests that the saturation of syn sites is causing protein “deactivation.” Previous biochemical and biophysical studies were focused on the identification of structural changes accompanying binding of cAMP to CRP, as well as on the determination what specific residues are involved in effecting observed transitions (reviewed in Ref. 1.Kolb A. Busby S. Buc H. Garges S. Adhya S. Annu. Rev. Biochem. 1993; 62: 749-795Crossref PubMed Google Scholar). However, none of those studies enabled identification of the mechanism, underlying the observed allosteric changes, in terms of KNF or MWC models. This paper presents the results of kinetic investigations of cAMP-induced conformational changes in CRP. Presented results provide evidence that allosteric “activation” of CRP at micromolar cAMP concentrations, occurs according to sequential (KNF) model, while conformational change observed at millimolar concentrations of cAMP, occurs according to concerted (MWC) model. 1,5-I-AENS, phenylmethylsulfonyl fluoride, EDTA, and Tris were purchased from Sigma. DTT, cAMP, and cGMP were either from Sigma or Fluka. The Fractogel EMD SO3− 650 (M) was from Merck; Q Sepharose Fast Flow and Sephadex G-25 were from Amersham Pharmacia Biotech. The nutrients for bacterial growth were either from Difco Laboratories or Life Sciences. All other chemicals were analytical grade products from POCh-Gliwice. All measurements were performed in buffers prepared in water purified by a Millipore system. CRP wild-type (wt) was isolated fromE. coli strain SA500 containing the plasmid pHA7, which encodes the crp gene (8.Aiba H. Fujimoto S. Ozaki N. Nucleic Acids Res. 1982; 10: 1345-1361Crossref PubMed Scopus (211) Google Scholar). The plasmid pHA7 was a generous gift from Dr. S. Garges. Tryptophan (Trp) at position 13 or 85 of CRP was replaced by phenylalanine with the use of pHA7 plasmid. The mutagenesis was performed using overlap extension method withPwo DNA polymerase. Plasmids encoding mutant crpgenes were introduced into E. coli strain M182Δcrp, kindly provided by Dr. S. Busby. Bacteria were grown on Terrific broth at 37 °C overnight, in a Biostat B fermentor from Braun, Germany, and then harvested by centrifugation. CRP wt and mutants were purified at 4 °C, as described previously (9.Małecki J. Wasylewski Z. Eur. J. Biochem. 1997; 243: 660-669Crossref PubMed Scopus (24) Google Scholar), with some important modifications. The cells were disrupted only by sonification using six 30-s pulse bursts, and the cell debris were removed by centrifugation. The crude extract was loaded onto a column filled with Fractogel EMD SO3−, equilibrated with buffer: 20 mm sodium phosphate, pH 6.8, 1 mm EDTA, 1 mm DTT, and 1 mm phenylmethylsulfonyl fluoride, and the column was washed overnight. Proteins were eluted from the column with a linear gradient of NaCl (0–1.0 m) in same buffer, and under such conditions CRP was eluted at about 0.7m NaCl. Fractions containing CRP were pooled, dialyzed against buffer: 50 mm Tris-HCl, pH 7.9 (at 20 °C), 100 mm KCl, 1 mm EDTA, 1 mm DTT, and 5% glycerol, and passed through a column filled with Q Sepharose, equilibrated with same buffer. The protein was collected in the void volume, dialyzed, and stored in aliquots at −20 °C. After this two-step procedure, the protein was highly pure (>97%), as judged by SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining. All measurements were performed in buffer A (50 mmTris-HCl, pH 7.9 (at 20 °C), 100 mm KCl, and 1 mm EDTA). Before measurements the protein aliquots stored at −20 °C were withdrawn, thawed on ice, and dialyzed extensively against buffer A to remove DTT and glycerol. Before measurements all samples were filtered through a microporous filter (0.45 μm) to remove all undissolved impurities. The concentration of protein, cyclic nucleotides, and fluorescence probe was determined by absorption spectroscopy using the following extinction coefficients: 40,800m−1 cm−1 at 278 nm for CRP wt dimer (6.Takahashi M. Blazy B. Baudras A. Biochemistry. 1980; 19: 5124-5130Crossref PubMed Scopus (98) Google Scholar), 14,650 m−1 cm−1 at 259 nm and 12,950 m−1 cm−1 at 254 nm for cAMP and cGMP (10.Merck Co Inc The Merck Index. 9th Ed. Merck & Co., Inc., Rahway, NJ1976: 353Google Scholar), respectively, and 6000m−1 cm−1 at 340 nm for AENS group (11.Hudson E.N. Weber G. Biochemistry. 1973; 12: 4154-4161Crossref PubMed Scopus (390) Google Scholar). Extinction coefficients of CRP mutants were determined using the method described elsewhere (12.Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5046) Google Scholar) and were 29,700m−1 cm−1 and 33,100m−1 cm−1 at 278 nm for W85F and W13F dimers, respectively. CRP wt was covalently labeled with 1,5-I-AENS using the procedure described elsewhere (13.Wu F.Y.-H. Nath K. Wu C.-W. Biochemistry. 1974; 13: 2567-2572Crossref PubMed Scopus (67) Google Scholar), with some minor modifications. Protein and label were mixed at molar ratio 1:20 and incubated at 4 °C in buffer A, overnight at dark. Labeled CRP was purified by Sephadex G-25 column and dialyzed extensively against buffer A. The stoichiometry of labeling was determined by absorption spectroscopy and was in the range of 2.2–2.6 mol of label/mol of CRP dimer. Kinetic measurements were performed at 20 °C (± 0.2 °C) using a stopped-flow spectrafluorimeter SX-17 MV from Applied Photophysics, United Kingdom. The dead time of mixing was determined to be less than 2 ms. Changes in the protein conformation induced by cAMP binding were monitored using two methods: 1) fluorescence intensity of AENS-CRP, observed through a green filter at wavelengths between 420 and 650 nm, after excitation at 340 nm and 2) fluorescence intensity of tryptophan (Trp) residues, observed through a cut-off filter at wavelengths >320 nm, after excitation at 295 nm. Trp fluorescence intensity was corrected for the inner filter effect, introduced by cAMP at the excitation wavelength, according to the following formula (14.Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983Crossref Google Scholar). Fcor=F×10P+ΔA2Equation 1 F and F cor are fluorescence intensity before and after the correction, whereas P and ΔA denote the initial sample absorption at the excitation wavelength and the change in absorption introduced by the ligand. In a typical series of experiments, the protein at fixed concentration was stopped-flow mixed with cAMP of various concentrations (mixing ratio 1:1). In experiments where AENS-CRP was used, its final concentration was 0.35 μm. In experiments where Trp fluorescence detection was used, protein final concentration was 2 μm. Between 10 and 14 kinetic traces were averaged routinely for each experiment. Such averaged kinetic traces were fit to a single exponential or a sum of such terms. F(t)=Aexp(−kt)+CEquation 2 F is the fluorescence intensity at time t,A and k are the amplitude and the observed rate constant, and C is the fluorescence at infinite time, respectively. The software for analysis of kinetic traces was from Applied Photophysics. At micromolar cAMP concentrations, CRP undergoes a conformational change according to sequential (KNF) model of allostery (see “Discussion”). This can be represented by the scheme shown by Reactions 1 and 2 and Equation 3. CRP+2cAMP ↔K1 CRPcAMP+cAMP ↔K2 CRPcAMP2 REACTION 1 CRPcAMP2 ↔k−ckc CRP*cAMP2 REACTION 2 KC=k−ckcEquation 3 K 1 and K 2 are intrinsic dissociation constants for binding of the first and the second cAMP molecule to high affinity (anti) sites, CRP and CRP* represent protein before and after the conformational change,KC is the equilibrium constant between these two forms, while kc and k-c are rate constants that describe the conformational change step. The observed rates (k obs) derived in kinetic experiments performed at micromolar cAMP concentrations can be fit to the following equation to yield the kinetic and thermodynamic parameters kc ,k -c, andKanti . kobs=k−c+kc[cAMP] 2Kanti2+2Kanti[cAMP]+[cAMP] 2Equation 4 For the simplicity of the analysis, the two high affinity sites were considered identical and independent and characterized by an average dissociation constant Kanti . The change in the fluorescence intensity, resulting from the conformational change of CRP occurring at micromolar cAMP concentrations, can be fit to the following equation to yield thermodynamic parameters K anti andKC . ΔFobs=ΔFmax[cAMP] 2Kanti2KC+2KantiKC[cAMP]+(1+KC)[cAMP] 2Equation 5 ΔF obs and ΔF max are the observed and the maximum change in fluorescence intensity in going from CRP-cAMP2 to CRP*-cAMP2, respectively. At millimolar cAMP concentrations, CRP undergoes a conformational change according to concerted (MWC) model of allostery (see “Discussion”), which can be represented by the scheme shown by Reactions 3 and 4 and Equation 6. P ↔koffkon P′ REACTION 3 K0=koffkonEquation 6 P′+2cAMP ↔K3 P′cAMP+cAMP ↔K4 P′cAMP2 REACTION 4P′ and P are the binding and non-binding form 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, whileK 3 and K 4 are intrinsic dissociation constants for binding of the first and the second cAMP molecule to low affinity (syn) sites. The observed rates (k obs) derived in kinetic experiments performed at millimolar cAMP concentrations can be fit to the following equation to yield the kinetic and thermodynamic parameters k on, k off, andKsyn . kobs=kon+koffKsyn2Ksyn2+2Ksyn[cAMP]+[cAMP] 2Equation 7 For simplicity of analysis, the two low affinity sites were considered identical and independent and characterized by an average dissociation constant Ksyn . The change in the fluorescence intensity, resulting from the conformational change of CRP occurring at millimolar cAMP concentrations, can be fit to the following equation to yield thermodynamic parameters Ksyn andK 0. ΔFobs=ΔFmax2Ksyn[cAMP]+[cAMP] 2Ksyn2(1+K0)+2Ksyn[cAMP]+[cAMP] 2Equation 8 ΔF obs and ΔF max are the observed and the maximum change in the fluorescence intensity in going from P to P′ form of protein, respectively. After excitation at 340 nm, the AENS-CRP emits a characteristic fluorescence with a maximum set at about 490 nm. Addition to the solution of 100 μm [cAMP] causes about 20% increase in the fluorescence intensity of AENS-CRP and concomitant blue shift in the emission maximum by about 10 nm. A further increase in [cAMP] to millimolar levels does not change the fluorescence characteristics of the labeled protein. When AENS-CRP is stopped-flow mixed with cAMP of various concentrations, a characteristic increase in the fluorescence intensity of the label is observed (Fig. 1). The obtained kinetic traces were fit to a single exponential or a sum of such terms (Equation 2). In all cases the data were satisfactorily described by a single exponential, while double exponential fitting did not improve the goodness of the fit. The rates obtained from fitting of AENS-CRP fluorescence detected kinetic traces to single exponential were plotted against the final cAMP concentration and the resulting graph is presented in Fig.2, while the corresponding amplitudes are presented in Fig. 3. As the total cAMP concentration increases, the observed rate (k obs) also increases. For ligand concentrations below 40 μm, an approximate linear dependence ofk obs on [cAMP] is observed. Above 40 μm [cAMP], the k obs deviates from linearity and approaches a constant value at very high (millimolar) concentrations of cAMP. Such behavior indicates a sequential two-step mechanism, where the process of conformational change is consecutive versus the ligand-binding step. Previous studies indicate that the association of cAMP to CRP is a very fast process (15.Wu C.-W. Wu F.Y.-H. Biochemistry. 1974; 13: 2573-2578Crossref PubMed Scopus (43) Google Scholar), suggesting that the observed change in the fluorescence intensity of AENS-CRP results from conformational change of the double liganded protein, i.e.: CRP-cAMP2(Equation 3 and Reactions 1 and 2).Figure 3Dependence of the amplitudes of cAMP-induced changes in the fluorescence intensity of AENS-CRP , on the final cAMP concentration, derived from kinetic (○) and equilibrium (■) measurements. The solid line denotes the best fit of data to Equation 5. The parameters of the fit are summarized in Table I.View Large Image Figure ViewerDownload Hi-res image Download (PPT) From the dependence of k obs on cAMP concentration, it is possible to evaluate the rate constants that describe the conformational change step and the average dissociation constant for cAMP binding to high affinity sites of CRP (Equation 4). The following values of the fitted parameters were obtained:kc = 9.7 ± 0.1 s−1,k-c = 0.31 ± 0.05 s−1, andKanti = 27.5 ± 1 μm (TableI). The above values enable to calculate the equilibrium constant between CRP-cAMP2 before and after the conformational change, which is KC = 0.032. Such low value of KC indicates that, in equilibrium, most of the double liganded CRP will undergo the conformational change.Table IKinetic and thermodynamic parameters describing cAMP binding to high affinity sites of CRP wtProteink ck −cK CK antis −1s −1μmCRP wt9.7 ± 0.1aObtained from rate-based analysis of kinetic data.0.31 ± 0.05aObtained from rate-based analysis of kinetic data.0.032 (0.029, 0.044)aObtained from rate-based analysis of kinetic data.27.5 ± 1.0aObtained from rate-based analysis of kinetic data.0.2 ± 0.09bObtained from amplitude-based analysis of kinetic data.9.6 ± 2.3bObtained from amplitude-based analysis of kinetic data.0.019 ± 0.015cObtained from analysis of equilibrium data.26.5 ± 11.6cObtained from analysis of equilibrium data.The values presented are derived from experiments conducted at 20 °C, in buffer A, pH 7.9, using AENS-CRP fluorescence detection. Kinetic and thermodynamic parameters are defined as described under “Experimental Procedures.” The error is the standard deviation of fitted parameters.a Obtained from rate-based analysis of kinetic data.b Obtained from amplitude-based analysis of kinetic data.c Obtained from analysis of equilibrium data. Open table in a new tab The values presented are derived from experiments conducted at 20 °C, in buffer A, pH 7.9, using AENS-CRP fluorescence detection. Kinetic and thermodynamic parameters are defined as described under “Experimental Procedures.” The error is the standard deviation of fitted parameters. The dependence of the amplitudes, associated with the observed rates, on the final [cAMP] is shown in Fig. 3. The presented binding curve saturates fully at about 100 μm [cAMP], with an apparent half-saturation point set at about 5.5 μm. This corresponds well with another binding curve (Fig. 3), which represents cAMP-induced changes in the fluorescence intensity of AENS-CRP, obtained from equilibrium studies. Both sets of data were fit to Equation 5, and the best-fit parameters are summarized in Table I. Presented results are in good agreement with values obtained from rate-based analysis. However, the analysis of the amplitude-based data gives a slightly lower value of the average dissociation constantKanti = 9.6 ± 2.5 μm and a higher value of the equilibrium constant KC = 0.2 ± 0.09, comparing to rate-based data analysis. When CRP is stopped-flow mixed with cAMP at concentrations below 300 μm, only very small change in the fluorescence intensity of Trp residues can be detected. However, when CRP is mixed with cAMP of higher concentrations, a characteristic increase in the fluorescence intensity of Trp residues is observed (Fig. 4). The obtained kinetic traces were fit to a single exponential or a sum of such terms (Equation 2). In all cases studied, the data were satisfactorily described by a single exponential, while double exponential fitting did not improve the goodness of the fit. The rates obtained from fitting of Trp fluorescence detected kinetic traces were plotted against the final [cAMP] and the resulting plot is presented in Fig. 5. The corresponding amplitudes are shown in Fig. 6. As the total cAMP concentration increases, the observed rate (k obs) decreases, reaching a constant value at very high ligand concentrations. Such behavior is indicative of displacement of an equilibrium between two conformational states of the protein, caused by the binding of ligand to one of these forms only (Equation 6 and Reactions 3 and 4).Figure 6Dependence of the amplitudes associated with cAMP-induced changes in Trp fluorescence intensity of CRP wt (○) and W13F mutant (■), on the final cAMP concentration. Thesolid line denotes the best fit of data to Equation 8, with the parameters of the fit summarized in TableII.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because the association of cAMP to CRP was shown to be a very fast process (15.Wu C.-W. Wu F.Y.-H. Biochemistry. 1974; 13: 2573-2578Crossref PubMed Scopus (43) Google Scholar), it can be assumed that P and P′ denote two interconvertible forms of CRP-cAMP2 complex (see “Discussion”). Moreover, as the observed changes occur at millimolar [cAMP], they must be associated with binding of cAMP to low affinity sites of CRP, but not with cAMP binding to high affinity sites. In order to evaluate the rate constants of the conformational change associated with P to P′ transition, as well as the average dissociation constant for cAMP binding to low affinity sites of CRP, the rate dependence from Fig. 5 was fit to Equation 7. The following values of the fitted parameters were obtained: k on = 28 ± 1.5 s−1, for the forward reaction,k off = 75.5 ± 3 s−1 for the backward reaction, and Ksyn = 2.0 ± 0.2 mm for the average dissociation constant of thesyn sites. From these values one can calculate the equilibrium constant between the two forms of CRP to be:K 0 = 2.7. All presented results are summarized in Table II.Table IIKinetic and thermodynamic parameters describing cAMP binding to low affinity sites of CRP wt and W13FProteink onk offK 0K syns −1s −1mmCRP wt28 ± 1.5aObtained from rate-based analysis.75.5 ± 3aObtained from rate-based analysis.2.7 (2.5, 3.0)aObtained from rate-based analysis.2.0 ± 0.2aObtained from rate-based analysis.28.5 ± 2bObtained from rate-based analysis, initial 200 μm [cAMP].85 ± 7.5bObtained from rate-based analysis, initial 200 μm [cAMP].3.0 (2.5, 3.5)bObtained from rate-based analysis, initial 200 μm [cAMP].1.8 ± 0.3bObtained from rate-based analysis, initial 200 μm [cAMP].4.4 ± 1.1cObtained from amplitude-based analysis.1.0 ± 0.2cObtained from amplitude-based analysis.CRP W13F18.2 ± 1.5aObtained from rate-based analysis.80.5 ± 4.4aObtained from rate-based analysis.4.4 (3.9, 5.1)aObtained from rate-based analysis.1.4 ± 0.2aObtained from rate-based analysis.6.4 ± 2.0cObtained from amplitude-based analysis.0.7 ± 0.15cObtained from amplitude-based analysis.The values presented are derived from experiments conducted at 20 °C, in buffer A, pH 7.9, using Trp fluorescence detection, with initial absence of cAMP, unless otherwise specified. Kinetic and thermodynamic parameters are defined as described under “Experimental Procedures.” The error is the standard deviation of fitted parameters.a Obtained from rate-based analysis.b Obtained from rate-based analysis, initial 200 μm [cAMP].c Obtained from amplitude-based analysis. Open table in a new tab The values presented are derived from experiments conducted at 20 °C, in buffer A, pH 7.9, using Trp fluorescence detection, with initial absence of cAMP, unless otherwise specified. Kinetic and thermodynamic parameters are defined as described under “Experimental Procedures.” The error is the standard deviation of fitted parameters. Fig. 6 shows the amplitudes obtained from Trp fluorescence-detected kinetic traces of cAMP binding to CRP. The resulting binding curve saturates at about 10 mm [cAMP], with a half-saturation point set at about 1.5 mm. These data were fit to Equation8, and the following values of the fitted parameters were obtained:Ksyn = 1.0 ± 0.2 mm andK 0 = 4.4 ± 1.1 (Table II). Trp fluorescence-detected stopped-flow kinetics was also employed to study cAMP binding to CRP-cAMP2 complex. In this case CRP was incubated at 200 μm [cAMP] for 1 h and subsequently stopped-flow mixed with cAMP of various concentrations. Additionally, in that case, the characteristic increase in the fluorescence intensity of Trp residues could be satisfactorily fit to a single exponential. The dependence of k obs on final cAMP concentration is virtually identical to that obtained for CRP in initial absence of the ligand (Fig. 5). When Equation 7 was used for fitting these data, the following values of the estimated parameters were obtained: k on = 28.5 ± 2.0 s−1, k off = 85 ± 7.5 s−1, and Ksyn = 1.8 ± 0.3 mm. From the values above, one can estimate the equilibrium constant between two interconvertible forms of CRP-cAMP2 to be K 0 = 3.0. This is exactly the same as obtained during kinetic experiments where unliganded CRP wt was used (Table II). Analogous fluorescence stopped-flow experiments using Trp fluorescence detection were performed using two single-tryptophan-containing CRP mutants: W85F and W13F. In case of CRP W85F, only very small decrease in intrinsic fluorescence intensity was observed at micromolar [cAMP]. However, a strong increase in Trp fluorescence at millimolar [cAMP] was observed in case of CRP W13F, and the obtained kinetic traces could be well described by a single-exponential. The dependence of k obs on cAMP concentration obtained for W13F mutant is shown in Fig. 5, while the corresponding amplitude dependence is presented in Fig. 6. Both the rate and amplitude dependences are similar to results obtained in case of CRP wt. The values of parameters: k on, k off,K 0, and Ksyn derived from fitting those data to Equations 7 and 8 are also quite similar between CRP wt and W13F mutant (Table II). The ultimate goal of conformational changes induced by cAMP binding to high affinity (anti) sites is to change CRP from a low affinity, nonspecific DNA-binding protein, to a transcription activator that binds DNA with high affinity and sequence specificity. According to present understanding of this phenomenon (2.Weber I.T. Steitz T.A. J. Mol. Biol. 1987; 198: 311-326Crossref PubMed Scopus (409) Google Scholar, 16.Garges S. Adhya S. J. Bacteriol. 1988; 170: 1417-1422Crossref PubMed Google Scholar, 17.Kim J. Adhya S. Garges S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9700-9704Crossref PubMed Scopus (79) Google Scholar), the cAMP-dependent switching of CRP into the active conformation involves at least three processes: subunit-subunit realignment, hinge reorientation between the domains, and α-helices' F protrusion. Results presented here provide evidence for conformational changes occurring within the HTH motif. Each CRP subunit contains three cysteine residues (8.Aiba H. Fujimoto S. Ozaki N. Nucleic Acids Res. 1982; 10: 1345-1361Crossref PubMed Scopus (211) Google Scholar); however, only Cys-178, which is located in the turn of the HTH motif, is solvent-accessible (4.Heyduk T. Lee J.C. Biochemistry. 19" @default.
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- W2019898190 title "Kinetic Studies of cAMP-induced Allosteric Changes in Cyclic AMP Receptor Protein from Escherichia coli" @default.
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