SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2000747979> ?p ?o ?g. }

Showing items 1 to 97 of 97 with 100 items per page.

W2000747979 endingPage "20006" @default.
W2000747979 startingPage "20001" @default.
W2000747979 abstract "Small angle neutron scattering (SANS) measurements were performed on solutions of cAMP receptor protein (CRP) and on solutions of the T127L,S128A double mutant of CRP (CRP*) in D2O K3PO4 buffer containing 0.5 m KCl, in the absence and presence of 3′,5′ cyclic adenosine monophosphate (cAMP). Energy-minimized structures of the CRP were calculated by minimization of the x-ray crystallographic structure of CRP in either the exclusively “closed” form where the α-helices of the carboxyl-terminal domain are folded close to the amino-terminal domain and in the exclusively “open” form where the α-helices of the carboxyl-terminal domain are folded away from the amino-terminal domain. Neutron scattering models show that the CRP SANS data follow closely the data curve predicted for unligated CRP in the open form, whereas the cAMP-ligated data are more in agreement with the data predicted for the minimized cAMP-ligated CRP structure in the closed form. Thus, it appears that CRP undergoes a conformational change from the open form to the closed form in solution upon ligation with cAMP. The SANS data from the CRP* and cAMP-ligated CRP* are coincidental, which implies that there is very little structural difference between the two species of CRP*. This is in agreement within vivo results, which show that whereas CRP activates transcription in the cell only in the presence of cAMP, CRP* activates transcription in the absence of cAMP, implying that CRP* is already in the correct conformation for the activation of transcription. Small angle neutron scattering (SANS) measurements were performed on solutions of cAMP receptor protein (CRP) and on solutions of the T127L,S128A double mutant of CRP (CRP*) in D2O K3PO4 buffer containing 0.5 m KCl, in the absence and presence of 3′,5′ cyclic adenosine monophosphate (cAMP). Energy-minimized structures of the CRP were calculated by minimization of the x-ray crystallographic structure of CRP in either the exclusively “closed” form where the α-helices of the carboxyl-terminal domain are folded close to the amino-terminal domain and in the exclusively “open” form where the α-helices of the carboxyl-terminal domain are folded away from the amino-terminal domain. Neutron scattering models show that the CRP SANS data follow closely the data curve predicted for unligated CRP in the open form, whereas the cAMP-ligated data are more in agreement with the data predicted for the minimized cAMP-ligated CRP structure in the closed form. Thus, it appears that CRP undergoes a conformational change from the open form to the closed form in solution upon ligation with cAMP. The SANS data from the CRP* and cAMP-ligated CRP* are coincidental, which implies that there is very little structural difference between the two species of CRP*. This is in agreement within vivo results, which show that whereas CRP activates transcription in the cell only in the presence of cAMP, CRP* activates transcription in the absence of cAMP, implying that CRP* is already in the correct conformation for the activation of transcription. The binding of 3′,5′ cyclic adenosine monophosphate (cAMP) to cAMP receptor protein (CRP) 1The abbreviations used are: CRPcAMP receptor proteinCRP*T127L,S128A mutant of CRPSANSsmall angle neutron scattering measurements.1The abbreviations used are: CRPcAMP receptor proteinCRP*T127L,S128A mutant of CRPSANSsmall angle neutron scattering measurements. activates the transcription of over 20 genes that code for the catabolite enzymes involved in carbohydrate metabolism inEscherichia coli. Results from NMR (1Hinds M.G. King R.W. Feeney J. Biochem. J. 1992; 287: 627-632Crossref PubMed Scopus (15) Google Scholar, 2Gronenborn A.M. Clore G.M. Biochemistry. 1982; 21: 4040-4047Crossref PubMed Scopus (57) Google Scholar), Raman spectroscopy (3DeGrazia H. Harman J.G. Tan G. Wartell R.M. Biochemistry. 1990; 29: 3557-3562Crossref PubMed Scopus (22) Google Scholar), proteolysis studies (4Cheng X. Kovac L. Lee J.C. Biochemistry. 1995; 34: 10816-10826Crossref PubMed Scopus (38) Google Scholar), small angle x-ray scattering measurements (5Kumar S.A. Murthy N.S. Krakow J.S. FEBS Lett. 1980; 109: 121-124Crossref PubMed Scopus (33) Google Scholar), and isothermal titration calorimetry (6Gorshkova I. Moore J.L. McKenney K.H. Schwarz F.P. J. Biol. Chem. 1995; 270: 21679-21683Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) indicate that upon binding to cAMP, CRP undergoes a conformational change to a conformation that promotes specific binding to the catabolite operons to activate transcription in the presence of RNA polymerase. The nature of this conformational change is not known, because only the structures of the cAMP-ligated CRP complex and of the DNA-cAMP-ligated CRP complex have been determined from x-ray crystallography studies (7Weber I.T. Steitz T.A. J. Mol. Biol. 1987; 198: 311-326Crossref PubMed Scopus (408) Google Scholar, 8McKay D.B. Steitz T.A. Nature. 1981; 290: 744-749Crossref PubMed Scopus (460) Google Scholar). cAMP receptor protein T127L,S128A mutant of CRP small angle neutron scattering measurements. cAMP receptor protein T127L,S128A mutant of CRP small angle neutron scattering measurements. CRP exists as a homodimer of 45,000 g mol−1 consisting of a β-pleated sheet amino-terminal domain, which contains the cAMP binding site and a carboxyl-terminal domain consisting of several α-helices that bind to specific DNA sequences. In the crystal phase, the cAMP-ligated CRP dimer is asymmetric where one monomer is in an “open” form in which the α-helices are swung out away from the amino-terminal domain and the other monomer is in a “closed” form in which the α-helices are swung in close to the amino-terminal domain (7Weber I.T. Steitz T.A. J. Mol. Biol. 1987; 198: 311-326Crossref PubMed Scopus (408) Google Scholar). The amino acid sequence connecting the two domains is, thus, called the “hinge” region. The x-ray crystal structure of the cAMP-ligated CRP dimer complexed with a 30-base pair DNA sequence is exclusively in the closed form (8McKay D.B. Steitz T.A. Nature. 1981; 290: 744-749Crossref PubMed Scopus (460) Google Scholar). Recent energy minimization computations of the two forms in solution show that the lowest energy conformation of the cAMP-ligated CRP dimer is exclusively the closed form in solution (9Garcia A.E. Harman J.G. Protein Sci. 1996; 5: 62-71Crossref PubMed Scopus (48) Google Scholar). The implication is that the asymmetry of the CRP dimer in the crystal lattice is due to crystal packing forces and that the structural change responsible for the activation of transcription is a change from the open to the closed form in solution. This is substantiated by NMR measurements on fluorinated derivatives of CRP, which imply that the conformational change involves the hinge region (1Hinds M.G. King R.W. Feeney J. Biochem. J. 1992; 287: 627-632Crossref PubMed Scopus (15) Google Scholar), and proton NMR measurements, which show that CRP in solution tightens up and becomes more rigid upon binding of cAMP (2Gronenborn A.M. Clore G.M. Biochemistry. 1982; 21: 4040-4047Crossref PubMed Scopus (57) Google Scholar). There is, however, evidence that the conformational change may not simply involve a transition from the open to the closed form of CRP upon binding to cAMP in solution. A comparison of the secondary structure of free CRP and cAMP-ligated CRP by Raman spectroscopy (3DeGrazia H. Harman J.G. Tan G. Wartell R.M. Biochemistry. 1990; 29: 3557-3562Crossref PubMed Scopus (22) Google Scholar) shows a structural shift from 44% α-helix, 28% β-strand, 18% turn, and 10% undefined in the free form to 37% α-helix, 33% β-strand, 17% turn, and 12% undefined in the ligated form, implying that the conformational change does not conserve the α-helical and β-strand structure as it would in a shift of the CRP dimer from the exclusively open to the closed form in solution. A conformational change involving alterations in the α-helical and β-strand structure of CRP is also implied by an empirical model of CRP secondary structure based on its sequence. This sequence-based empirical model predicts α-helices in unligated CRP in place of the β-strands in the amino-terminal domain of the x-ray crystallographic structure of the cAMP-ligated CRP complex (10Kypr J. Mrazek J. Biochem. Biophys. Res. Commun. 1985; 131: 780-785Crossref PubMed Scopus (5) Google Scholar). In addition to the contradictory evidence concerning the nature of the conformational change in CRP, there is also experimental evidence that a cAMP-induced conformational change is not always necessary for the activation of transcription. For example, in vivotranscription was observed to be activated even in the absence of cAMP by a mutant of CRP where two amino acid residues have been replaced in the amino-terminal domain of CRP, T127L and S128A (CRP*) (11Moore J.L. The Mechanism of Activation of the Escherichia coli Cyclic AMP Receptor Protein by Cyclic AMP. Ph.D. thesis. The George Washington University, Washington, D. C.1993Google Scholar). This implies that CRP* is already in the transcriptionally activated conformation and that cAMP binding is unlikely to initiate a conformational change. Small angle neutron scattering (SANS) measurements have been employed to determine the conformation and size of proteins in solution (12Timmins P.A. Zacci G. Eur. Biophys. J. 1988; 15: 257-268Crossref PubMed Scopus (48) Google Scholar). Using D2O as the solvent to maximize the difference in neutron scattering between the solvent and the protein, the conformation of the protein can be determined from the dependence of the scattered neutron intensity on the scattering angle and the size of the protein in terms of the radius of gyration (Rg) can be determined from the scattered neutron intensity near zero scattering angle (13Guinier A. Fournet G. Small-Angle Scattering of X-rays. John Wiley & Sons, New York1955Google Scholar). In addition, the SANS data can be compared with scattered neutron intensity curves generated from the energy-minimized crystallographic x-ray structure of the protein to determine in more detail the conformation of the protein in solution. In this investigation, SANS measurements were performed on solutions of CRP in the absence and presence of cAMP and compared with model scattered intensity curves calculated from the energy-minimized x-ray structures of CRP dimer in exclusively the closed and in exclusively the open forms. For comparison, SANS measurements were also performed on solutions of CRP* in the absence and presence of cAMP. The characterization and mutagenesis of the wild type and mutant of CRP have been described previously by Moore (11Moore J.L. The Mechanism of Activation of the Escherichia coli Cyclic AMP Receptor Protein by Cyclic AMP. Ph.D. thesis. The George Washington University, Washington, D. C.1993Google Scholar). A New Brunswick Fermentor Scientific, Inc. SF116 was also used in place of the 5-liter shaker flasks for preparations of CRP and CRP*. An Amersham Pharmacia Biotech Phast SDS-electrophoresis gel with 0.1m Tris-HCl buffer (pH 7.0) containing 0.2 mass % bromphenol blue and 20 volume % glycerol was run to determine the purity of the protein preparations. A protein analysis was also performed by comparing the absorbance at 562 nm of samples of the solution to those of bovine serum albumin solutions after adding 4 mass % CuSO4 and bicinchoninic acid reagent from Pierce. The protein solutions were dialyzed at 4 °C in 50 mmpotassium phosphate-potassium hydroxide buffer (pH = 7.0) containing 0.2 mm sodium EDTA, 0.2 mmdithiothreitol, 0.5 mm KCl, and 5 volume % glycerol (phosphate buffer) in D2O with two changes in the buffer solution. The concentrations of the CRP and the mutant were determined by UV absorption spectroscopy using an extinction coefficient of 3.5 × 104 cm liter mol−1 at 280 nm (14Ghosaini L.R. Brown A.M. Sturtevant J.M. Biochemistry. 1988; 27: 5257-5261Crossref PubMed Scopus (99) Google Scholar). The ligand solutions were made up by dissolving known masses of the sodium salt of the cyclic nucleotide in the D2O buffer. The sodium salt of cAMP, potassium phosphate, NaOH, EDTA, dithiothreitol, glycerol, Tris, HCl, bromphenol blue, and KCl were reagent grade from Sigma. The SANS measurements were performed on the NG3 30 m SANS instrument at the National Institute of Standards and Technology Center for Neutron Research in Gaithersburg, MD (15Hammouda B. Krueger S. Glinka C.J. J. Res. Natl. Inst. Stand. Technol. 1993; 98: 31-46Crossref PubMed Scopus (96) Google Scholar). Unligated CRP solutions were measured at 0.036 and 0.096 mm and unligated CRP* at 0.049 and 0.138 mm in the D2O solvent. CRP was also measured at both concentrations in the presence of 0.270 and 17.1 mm cAMP, whereas CRP* solutions at both concentrations were measured in the presence of 17.1 mm cAMP. To limit aggregation effects, the lower concentration solutions were used to obtain the data at the lower scattering angles where information about the overall size of the protein is obtained, whereas the higher concentration solutions were used to obtain the higher angle data, which provide information about the shape of the protein. In the higher angle scattering region, aggregation effects are much smaller than in the lower angle region, but the scattered intensity is also much lower, so a higher concentration of CRP and CRP* in solution is needed in the higher angle region. Data were taken at a wavelength λ = 5 Å, with a spread Δλ/λ = 0.34 for the CRP samples and Δλ/λ = 0.15 for the CRP* samples. A sample-to-detector distance of 5.5 m and a source-to-sample distance of 7.02 m were used for the 0.036 and 0.049 mm samples to obtain the lower angle data. The 0.096 and 0.138 mm samples were measured at a sample-to-detector distance of 1.5 m and a source-to-sample distance of 3.92 m to obtain the higher angle data. The source and sample apertures were 5.0 and 1.27 cm, respectively. Neutrons were detected on a 64.0 × 64.0-cm two-dimensional position-sensitive detector with 1.0-cm resolution. The center of the detector was offset by 20.0 cm at both sample-to-detector distance positions to provide a large overlap between the two configurations and to measure to a large enough scattering angle to allow an accurate determination of the incoherent background scattering due to hydrogen in the sample. The scattered intensity, I, was corrected for solvent absorbance and scattering on a pixel-by-pixel basis using the relation, Icor=Isample−Tsample/TsolventIsolventEquation 1 where Tsample andTsolvent are the measured transmissions of neutrons through the sample and solvent, respectively. The corrected data were placed on an absolute scale by calibration against the scattering from a silica gel standard sample and then radially averaged to obtain I(Q) as a function of Q, where Q = 4πsinθ/λ, and 2θ is the scattering angle. The radially averaged data exhibit a distinct flat region beyondQ = 0.35 Å−1 due to the incoherent scattering from hydrogen in the samples. This background scattering was subtracted from the data taken at both instrument configurations, normalizing for the difference in the protein concentrations. The background-subtracted data for both configurations were simultaneously corrected (desmeared) for instrument resolution effects, and Fourier transformed to obtain the distance distribution function,P(r), using two different methods: the Svergun method (16Svergun D.I. Semenyuk A.V. Feigin L.A. Acta Crystallogr. Sec. A. 1988; 44: 244-250Crossref Scopus (271) Google Scholar), as implemented in the program GNOM (17Semenyuk A.V. Svergun D.I. J. Appl. Crystallogr. 1991; 24: 537-540Crossref Scopus (564) Google Scholar), and the Glatter method (18Glatter O. J. Appl. Crystallogr. 1977; 10: 415-421Crossref Google Scholar), as implemented in the program GLATTER, with resolution effects incorporated by Skov Pederson et al. (19Skov Pederson J. Posselt D. Mortensen K. J. Appl. Crystallogr. 1990; 23: 321-333Crossref Google Scholar). Both methods assume the scattering results from a dilute, monodispersed solution of globular particles. In addition, it is assumed that the particles have a sharp boundary such that there is a maximum diameter,Dmax, beyond which there is no significant scattering mass. The desmeared scattered intensity,I(Q), is related to P(r) by the equation, I(Q)=4πV0∫0DmaxP(r)sin(Qr)/Qr drEquation 2 where the two conditions, P(0) = 0 andP(2r ≥ Dmax) = 0, are satisfied. 4πP(r) is defined as the number of distances, r, between scattering centers within the particle. The I(Q) versus Qdata taken at both instrument configurations are combined in the desmearing process, and I(Q) versus Q data were obtained over a usable range 0.012 ≤Q ≤ 0.3 Å−1. The results from the two desmearing methods were averaged to produce the final desmearedI(Q) versus Q data andP(r) versus r data. In the small angle limit, the scattered intensity can be approximated by the Guinier equation (13Guinier A. Fournet G. Small-Angle Scattering of X-rays. John Wiley & Sons, New York1955Google Scholar), I(Q)=I(0)exp−Q2Rg2/3Equation 3 where I(0) is the scattered intensity atQ = 0. The radius of gyration of the particle,Rg, is defined as the mean square distance from the center of scattering mass. A plot of ln(I)versus Q 2 should contain a linear region with slope Rg2/3 and intercept ln(I(0)). Thus, Rg can be obtained directly from the SANS measurements. The range of validity for the Guinier approximation depends upon the shape of the particle. However, Guinier fits are generally made in a Q range centered about the relation, QRg ∼ 1.0. Details of the data desmearing procedure are illustrated in Fig.1, which shows the smearedI(Q) versus Q data for the unligated CRP sample measured in D2O solvent. Thedotted line is the best fit to the data using the Glatter (18Glatter O. J. Appl. Crystallogr. 1977; 10: 415-421Crossref Google Scholar, 19Skov Pederson J. Posselt D. Mortensen K. J. Appl. Crystallogr. 1990; 23: 321-333Crossref Google Scholar) method, in which the data are represented by a series of B-splines. The data were fitted several times using different estimates for Dmax. The fit that resulted in the smoothestP(r) function for which theDmax value was clearly not too large, and for which the calculated I(Q) still fitted well to the experimental data, was chosen as the best solution. Dmax values of 65 ± 5 Å in Equation 2proved to fit the smeared data best in all cases. The “x” points show the desmeared data resulting from the dotted line fit. Errors on the desmeared data were determined by averaging the best-fit results from both the Svergun (16Svergun D.I. Semenyuk A.V. Feigin L.A. Acta Crystallogr. Sec. A. 1988; 44: 244-250Crossref Scopus (271) Google Scholar, 17Semenyuk A.V. Svergun D.I. J. Appl. Crystallogr. 1991; 24: 537-540Crossref Scopus (564) Google Scholar) and Glatter (18Glatter O. J. Appl. Crystallogr. 1977; 10: 415-421Crossref Google Scholar, 19Skov Pederson J. Posselt D. Mortensen K. J. Appl. Crystallogr. 1990; 23: 321-333Crossref Google Scholar) methods. The I(Q) versus Q data obtained from SANS measurements can be compared with that calculated from the energy-minimized x-ray crystallographic structure using a Monte Carlo technique (20Heidorn D.B. Trewhella J. Biochemistry. 1987; 27: 909-915Crossref Scopus (279) Google Scholar). Since hydrogen atoms are not included in the x-ray structure, each amino acid is better represented by a homogeneous sphere positioned according to its α-carbon coordinates. The size and neutron scattering strength of each sphere are calculated from the known volume and chemical composition of the amino acid, respectively, thus taking into account the contribution of hydrogen. Once a sphere has been drawn around each amino acid, a box is drawn around the entire molecule, with the option to include a region of bound water up to 5 Å thick. The bulk solvent can be any H2O/D2O mixture, and the protein can be hydrogenated or deuterated. Points are then generated at random within the box. The distance distribution function,P(r), for the total volume is calculated directly in real space by making a histogram representing the distances between all possible pairs of points, weighted according to the product of the neutron scattering strengths at each point. Rgis calculated directly from the second moment ofP(r) and I(Q) is found using Equation 2. The x-ray crystallographic structure of the cAMP-ligated CRP dimer shows that it can exist in an open and closed form in the crystal phase. The CRP dimers were generated from the Brookhaven Protein Data Bank file 3GAP.pdb exclusively in either the closed or open form by superimposing a copy of the monomer in the closed from on the monomer in the open form and vice versa (20Heidorn D.B. Trewhella J. Biochemistry. 1987; 27: 909-915Crossref Scopus (279) Google Scholar). The energetics of the structures were then minimized using the steepest descent method for 1000 iterations, after which the conjugate gradient method was used until convergence was achieved (maximum derivative less than 0.04 kJ mol−1). Computational results were obtained using software programs from Biosym/MSI of San Diego. Energy minimizations, using the AMBER forcefield, were done with the Discover program, and structure analysis and comparison were performed with the DeCipher program. Energy-minimized structures for the unliganded CRP were generated in the same manner, using the cAMP-ligated x-ray structure, with the cAMP removed, as the starting structure. The desmeared I(Q) versus Q data for the CRP and the cAMP-ligated CRP solutions are plotted in Fig. 2. It should be emphasized that at the levels of 0.036 mm CRP and 0.270 mm cAMP concentrations, the CRP is only about 90% ligated, so that the scattering would contain some contribution from unligated CRP. At higher cAMP concentrations of approximately 17.1 mm, where the CRP would be fully ligated (21Moore J.L. Gorshkova I.I. Brown J.W. McKenney K.H. Schwarz F.P. J. Biol. Chem. 1996; 271: 21273-21278Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), aggregation was observed in the CRP solutions as indicated by a sharp upward inflection of the neutron scattering intensity near Q = 0 and, thus, these data were not analyzed. The scattering curve for CRP in the presence of cAMP deviates significantly as shown in Fig. 2 from that of the unligated CRP above Q = 0.15 Å−1. This deviation results from a conformational change in CRP upon ligation with cAMP and is in contrast to the coincidence of the desmeared SANS data from solutions of CRP* in the absence and presence of 17.1 mm cAMP shown in Fig.3. (Aggregation at this cAMP concentration was not observed for the CRP* solutions.) The nearly coincidental curves in Fig. 3 show that CRP*, contrary to CRP, undergoes very little conformational change in the presence of cAMP, as implied by the in vivo activation of transcription by CRP* in the absence of cAMP.Figure 3Desmeared SANS intensity versus Q from 0.049 and 0.138 mm unligated CRP* (•) and CRP* + 17.1 mm cAMP (■) in D2O solvent.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The radii of gyration calculated from the second moment of the distance distribution function, P(r), using all of the desmeared data in Figs. 2 and 3 are presented in TableI. For comparison, Table I also lists the radii of gyration obtained by averaging the results from several Guinier fits over the Q range limited to 0.02 ≤Q ≤ 0.08 Å−1 for unligated and ligated CRP and CRP*. The radii of gyration calculated by the two methods are essentially the same for the unligated and cAMP-ligated CRP* solutions (approximately 22.3 ± 0.3 Å), indicating again virtually no difference between the size of CRP* and cAMP-ligated CRP*. This is in contrast with the results from the CRP solutions, using the two methods, which exhibit a consistent shrinkage of about 1.0 Å upon cAMP ligation. The slightly larger Rg values determined for CRP from the Guinier fits compared with those determine from the 2nd moment of P(r) may be due to the effect of some aggregation in the sample on the Guinier-derivedRg. The Rg values calculated from P(r) are not significantly affected by small deviations in the scattering curve due to aggregation effects at small Q, where the Guinier-derivedRg values were determined.Table IComparison of the Radii of Gyration (Rg) for CRP and CRPSampleRg from Guinier fitRg from 2nd moment of P(r)ExperimentalCalculated open formaCalculated from the energy-minimized x-ray structures of CRP.Calculated closed formaCalculated from the energy-minimized x-ray structures of CRP.ÅÅUnligated CRP23.0 ± 0.322.0 ± 0.220.5 ± 0.418.7 ± 0.3cAMP-ligated CRP22.1 ± 0.321.6 ± 0.219.2 ± 0.319.2 ± 0.3Unligated CRP*22.6 ± 0.322.2 ± 0.2cAMP-ligated CRP*22.9 ± 0.322.3 ± 0.2a Calculated from the energy-minimized x-ray structures of CRP. Open table in a new tab The desmeared data in Fig. 2 are compared in Fig.4 to model scattering curves calculated from their corresponding energy-minimized x-ray structures, using 2000 randomly placed points in the volume occupied by the molecule. Three iterations were performed for each of four structures, i.e.unligated CRP dimer in the exclusively open and closed forms, and cAMP-ligated CRP dimer in the exclusively open and closed forms. The final model curves represent the average of the three iterations. The unligated CRP scattered intensity, along with the model intensities calculated from the energy-minimized x-ray structures of CRP with cAMP removed, are plotted in Fig. 4 A as a function of the dimensionless parameter, Q·Rg(QRg). Plotting the curves in this manner maximizes the differences in structure due to changes in overall shape, while minimizing changes in Rg. In Fig.4 A, the experimental desmeared SANS data fit very closely the model curve for unligated CRP exclusively in the open form. The close agreement between the desmeared SANS data and the model curve of CRP in the open form indicates that the open form of CRP is the preferred conformation of unligated CRP in solution. This is further substantiated by the radii of gyration obtained from the second moment of P(r) for the energy-minimized x-ray structures listed in Table I. The Rg value from the desmeared unligated CRP data is 22.0 ± 0.2 Å, which compares more favorably with 20.5 ± 0.4 Å for the minimized, exclusively open form of CRP than with 18.7 ± 0.3 Å for the minimized, exclusively closed form of CRP. The desmeared scattered intensity for cAMP-ligated CRP is plotted in Fig. 4 B as a function of QRg, along with the model intensities obtained from the energy-minimized x-ray structures of cAMP-ligated CRP. The model curves for the minimized exclusively closed and open forms of the cAMP-ligated CRP are less separated than the corresponding model curves for unligated CRP in Fig.4 A and have Rg values of approximately 19.2 ± 0.3 Å in each case, compared withRg = 21.6 ± 0.2 Å for the desmeared data in Table I. However, the desmeared data appear to follow more closely the model curve calculated from the minimized structure of cAMP-ligated CRP dimer exclusively in the closed form. The SANS results show that the conformational change that occurs in CRP upon ligation with cAMP can well be described as a change from the open to the more compact closed form of CRP. This is further evident in the distance distribution functions, P(r), plotted for unligated and cAMP-ligated CRP in Fig.5 A. In the presence of cAMP, the peak shifts slightly toward smaller r values, indicating that cAMP-ligated CRP is more compact than nonligated CRP. Fig.5 B shows the P(r) functions obtained from the energy-minimized x-ray structures that fit most closely to the data. The distributions look remarkably similar to those in Fig.5 A, with the peak in the distribution for the cAMP-ligated CRP in the closed form occurring at a lower value than that for the nonligated CRP in the open form. The SANS results show that ligation of CRP by cAMP apparently induces a conformational change in CRP from the exclusively open form in solution to the more contracted closed form, whereas CRP* remains essentially unchanged in the presence of cAMP in solution. It should be emphasized that the energy-minimized open form of unligated CRP is based on energy minimization of the x-ray crystal structure of cAMP-ligated CRP, exclusively in the open form, with cAMP removed, because the x-ray crystal structure of unligated CRP is unavailable. The good agreement between this conformation of unligated CRP and the experimental SANS curve does imply that this energy-minimized open form may indeed be the stable conformation of unligated CRP in solution. These results agree with NMR measurements, which show a tightening up of CRP into a more rigid protein upon cAMP binding (2Gronenborn A.M. Clore G.M. Biochemistry. 1982; 21: 4040-4047Crossref PubMed Scopus (57) Google Scholar). The magnitude of the change of approximately 1 Å in the radius of gyration also agrees with the 0.7 ± 0.1 Å shrinkage observed in the Stokes radius of CRP upon ligation with cAMP (22Heyduk E. Heyduk T. Lee J.C. J. Biol. Chem. 1992; 267: 3200-3204Abstract Full Text PDF PubMed Google Scholar). Although the change in CRP is in agreement with earlier small angle x-ray scattering measurements, which also indicate a contraction in the shape of CRP upon binding of cAMP, the x-ray measurements exhibited a larger change inRg from 29 Å for unligated CRP to 25 Å for the cAMP-ligated CRP complex (5Kumar S.A. Murthy N.S. Krakow J.S. FEBS Lett. 1980; 109: 121-124Crossref PubMed Scopus (33) Google Scholar). Calculations on the energy-minimized unligated and ligated CRP structures show thatRg would contract only from 20.5 Å for the open form of CRP to 19.2 Å for the closed form of the complex. The x-ray scattering results show not only a large change inRg, but also an absoluteRg value 6–9 Å larger than could be calculated from the x-ray crystallographic structures. The presence of aggregates in the small angle x-ray scattering samples could easily account for the higher Rg values and larger relative change in Rg in the presence of cAMP. The change to the closed form for the cAMP-ligated CRP complexes in solution also agrees with molecular dynamics simulations on the cAMP-ligated CRP complex, which predict that the closed form is the more energetically stable form of this complex in solution (9Garcia A.E. Harman J.G. Protein Sci. 1996; 5: 62-71Crossref PubMed Scopus (48) Google Scholar). In addition, the x-ray crystallographic structure of the cAMP-ligated CRP complexed with DNA shows that the cAMP-ligated CRP exists exclusively in the closed form for this ternary complex (8McKay D.B. Steitz T.A. Nature. 1981; 290: 744-749Crossref PubMed Scopus (460) Google Scholar). Lack of growth for CRP crystals in the absence of cAMP may arise from the CRP in solution being exclusively in the open form. The energy-minimized structures for unligated CRP in the open form and cAMP-ligated CRP in the closed form are shown in Fig.6, which illustrates the small, but significant, structural differences between the two forms. A model proposed by Garges and Adhya (23Garges S. Adhya S. J. Bacteriol. 1988; 170: 1417-1422Crossref PubMed Google Scholar) for the conformational change of CRP upon cAMP binding involves four structural changes in CRP: 1) a change in the orientation of the D α-helix (Fig. 6), 2) an interaction between the cAMP and DNA binding domains, 3) re-alignment of the CRP monomers, and 4) more importantly, the “pushing away” of the DNA-binding F α-helices (Fig. 6) from the protein. These changes also allow the two F α-helices to bind into two adjacent grooves of DNA. The changes described by the model with respect to the interaction between the cAMP and DNA binding domains and a change in the distance between the DNA-binding F α-helices are also shown in Fig. 6 in the transition from the unligated open form of CRP (Fig. 6 A) to the ligated closed form of CRP (Fig. 6 B). The distance between the F α-helices in the closed form is more conducive to DNA binding as evident by the cAMP-CRP-DNA crystal structure, which is exclusively in the closed form. Thus, the changes described in this model support the SANS results, which show a conformational change from unligated CRP in exclusively the open form to the closed form upon cAMP ligation in solution. The lack of significant changes in the CRP* scattering curves upon complexation with cAMP implies that CRP* undergoes little, or possibly no, structural change in the presence of cAMP. This is in agreement with the observation that transcription is activated in the absence of cAMP in cells which contain CRP* (11Moore J.L. The Mechanism of Activation of the Escherichia coli Cyclic AMP Receptor Protein by Cyclic AMP. Ph.D. thesis. The George Washington University, Washington, D. C.1993Google Scholar), implying that unligated CRP* is already in the correct conformation for binding of the DNA. To substantiate that this conformation is the closed form, fits of the energy-minimized structure of CRP* to the SANS data must await the determination of a good starting structure for CRP* from x-ray crystallography." @default.
W2000747979 created "2016-06-24" @default.
W2000747979 creator A5030363413 @default.
W2000747979 creator A5044677762 @default.
W2000747979 creator A5063841415 @default.
W2000747979 creator A5064960135 @default.
W2000747979 creator A5076403584 @default.
W2000747979 creator A5084382524 @default.
W2000747979 date "1998-08-01" @default.
W2000747979 modified "2023-09-27" @default.
W2000747979 title "Determination of the Conformations of cAMP Receptor Protein and Its T127L,S128A Mutant with and without cAMP from Small Angle Neutron Scattering Measurements" @default.
W2000747979 cites W1540297453 @default.
W2000747979 cites W1560062702 @default.
W2000747979 cites W1978517242 @default.
W2000747979 cites W1981262848 @default.
W2000747979 cites W1982602765 @default.
W2000747979 cites W1983138565 @default.
W2000747979 cites W1992191979 @default.
W2000747979 cites W1999219397 @default.
W2000747979 cites W2005586626 @default.
W2000747979 cites W2025461272 @default.
W2000747979 cites W2027383974 @default.
W2000747979 cites W2038960066 @default.
W2000747979 cites W2045102512 @default.
W2000747979 cites W2047891873 @default.
W2000747979 cites W2053591536 @default.
W2000747979 cites W2062387389 @default.
W2000747979 cites W2078546091 @default.
W2000747979 cites W2082958294 @default.
W2000747979 cites W2093876265 @default.
W2000747979 cites W2094253502 @default.
W2000747979 cites W2271189536 @default.
W2000747979 doi "https://doi.org/10.1074/jbc.273.32.20001" @default.
W2000747979 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9685337" @default.
W2000747979 hasPublicationYear "1998" @default.
W2000747979 type Work @default.
W2000747979 sameAs 2000747979 @default.
W2000747979 citedByCount "35" @default.
W2000747979 countsByYear W20007479792012 @default.
W2000747979 countsByYear W20007479792013 @default.
W2000747979 countsByYear W20007479792014 @default.
W2000747979 countsByYear W20007479792015 @default.
W2000747979 countsByYear W20007479792016 @default.
W2000747979 countsByYear W20007479792019 @default.
W2000747979 crossrefType "journal-article" @default.
W2000747979 hasAuthorship W2000747979A5030363413 @default.
W2000747979 hasAuthorship W2000747979A5044677762 @default.
W2000747979 hasAuthorship W2000747979A5063841415 @default.
W2000747979 hasAuthorship W2000747979A5064960135 @default.
W2000747979 hasAuthorship W2000747979A5076403584 @default.
W2000747979 hasAuthorship W2000747979A5084382524 @default.
W2000747979 hasBestOaLocation W20007479791 @default.
W2000747979 hasConcept C104015590 @default.
W2000747979 hasConcept C104317684 @default.
W2000747979 hasConcept C121332964 @default.
W2000747979 hasConcept C12554922 @default.
W2000747979 hasConcept C132841051 @default.
W2000747979 hasConcept C143065580 @default.
W2000747979 hasConcept C152568617 @default.
W2000747979 hasConcept C170493617 @default.
W2000747979 hasConcept C185544564 @default.
W2000747979 hasConcept C185592680 @default.
W2000747979 hasConcept C55493867 @default.
W2000747979 hasConcept C86803240 @default.
W2000747979 hasConceptScore W2000747979C104015590 @default.
W2000747979 hasConceptScore W2000747979C104317684 @default.
W2000747979 hasConceptScore W2000747979C121332964 @default.
W2000747979 hasConceptScore W2000747979C12554922 @default.
W2000747979 hasConceptScore W2000747979C132841051 @default.
W2000747979 hasConceptScore W2000747979C143065580 @default.
W2000747979 hasConceptScore W2000747979C152568617 @default.
W2000747979 hasConceptScore W2000747979C170493617 @default.
W2000747979 hasConceptScore W2000747979C185544564 @default.
W2000747979 hasConceptScore W2000747979C185592680 @default.
W2000747979 hasConceptScore W2000747979C55493867 @default.
W2000747979 hasConceptScore W2000747979C86803240 @default.
W2000747979 hasIssue "32" @default.
W2000747979 hasLocation W20007479791 @default.
W2000747979 hasOpenAccess W2000747979 @default.
W2000747979 hasPrimaryLocation W20007479791 @default.
W2000747979 hasRelatedWork W1992856627 @default.
W2000747979 hasRelatedWork W2014873921 @default.
W2000747979 hasRelatedWork W20165201 @default.
W2000747979 hasRelatedWork W2066047096 @default.
W2000747979 hasRelatedWork W2923921346 @default.
W2000747979 hasRelatedWork W3104938576 @default.
W2000747979 hasRelatedWork W3133884853 @default.
W2000747979 hasRelatedWork W3152828946 @default.
W2000747979 hasRelatedWork W4313244371 @default.
W2000747979 hasRelatedWork W2123429877 @default.
W2000747979 hasVolume "273" @default.
W2000747979 isParatext "false" @default.
W2000747979 isRetracted "false" @default.
W2000747979 magId "2000747979" @default.
W2000747979 workType "article" @default.