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• W2050179344 abstract "Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ABC protein superfamily. Phosphorylation of a regulatory domain of this protein is a prerequisite for activity. We analyzed the effect of protein kinase A (PKA) phosphorylation on the structure of purified and reconstituted CFTR protein. 1H/2H exchange monitored by attenuated total reflection Fourier transform IR spectroscopy demonstrates that CFTR is highly accessible to aqueous medium. Phosphorylation of the regulatory (R) domain by PKA further increases this accessibility. More specifically, fluorescence quenching of cytosolic tryptophan residues revealed that the accessibility of the cytoplasmic part of the protein is modified by phosphorylation. Moreover, the combination of polarized IR spectroscopy with 1H/2H exchange suggested an increase of the accessibility of the transmembrane domains of CFTR. This suggests that CFTR phosphorylation can induce a large conformational change that could correspond either to a displacement of the R domain or to long range conformational changes transmitted from the phosphorylation sites to the nucleotide binding domains and the transmembrane segments. Such structural changes may provide better access for the solutes to the nucleotide binding domains and the ion binding site. Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ABC protein superfamily. Phosphorylation of a regulatory domain of this protein is a prerequisite for activity. We analyzed the effect of protein kinase A (PKA) phosphorylation on the structure of purified and reconstituted CFTR protein. 1H/2H exchange monitored by attenuated total reflection Fourier transform IR spectroscopy demonstrates that CFTR is highly accessible to aqueous medium. Phosphorylation of the regulatory (R) domain by PKA further increases this accessibility. More specifically, fluorescence quenching of cytosolic tryptophan residues revealed that the accessibility of the cytoplasmic part of the protein is modified by phosphorylation. Moreover, the combination of polarized IR spectroscopy with 1H/2H exchange suggested an increase of the accessibility of the transmembrane domains of CFTR. This suggests that CFTR phosphorylation can induce a large conformational change that could correspond either to a displacement of the R domain or to long range conformational changes transmitted from the phosphorylation sites to the nucleotide binding domains and the transmembrane segments. Such structural changes may provide better access for the solutes to the nucleotide binding domains and the ion binding site. Cystic fibrosis is caused by a mutation in the membrane chloride channel CFTR 1The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorABCATP binding cassetteATR-FTIRattenuated total reflection-Fourier transform IR spectroscopyPKAprotein kinase ANBDnucleotide binding domainPFOpentadecafluorooctanoic acid.1The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorABCATP binding cassetteATR-FTIRattenuated total reflection-Fourier transform IR spectroscopyPKAprotein kinase ANBDnucleotide binding domainPFOpentadecafluorooctanoic acid. (1Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5811) Google Scholar). CFTR is a member of the ABC superfamily. As the other members of this family, CFTR contains two transmembrane domains and two nucleotide binding domains (NBD) responsible for ATP hydrolysis. In addition to the common ABC structure, CFTR possesses an R domain (regulatory domain) that contains several consensus phosphorylation sites. Phosphorylation of this domain followed by ATP binding and hydrolysis by the NBDs is necessary to induce chloride permeability (2Li C. Ramjeesingh M. Wang W. Garami E. Hewryk M. Lee D. Rommens J.M. Galley K. Bear C.E. J. Biol. Chem. 1996; 271: 28463-28468Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 3Bear C.E. Li C.H. Kartner N. Bridges R.J. Jensen T.J. Ramjeesingh M. Riordan J.R. Cell. 1992; 68: 809-818Abstract Full Text PDF PubMed Scopus (770) Google Scholar). Yet, an alternative activation mode has been proposed recently, where glutamate induces chloride channel activity in the absence of PKA and ATP (4Reddy M.M. Quinton P.M. Nature. 2003; 423: 756-760Crossref PubMed Scopus (93) Google Scholar), but it still needs further investigation. cystic fibrosis transmembrane conductance regulator ATP binding cassette attenuated total reflection-Fourier transform IR spectroscopy protein kinase A nucleotide binding domain pentadecafluorooctanoic acid. cystic fibrosis transmembrane conductance regulator ATP binding cassette attenuated total reflection-Fourier transform IR spectroscopy protein kinase A nucleotide binding domain pentadecafluorooctanoic acid. CFTR contains 10 dibasic (R(R/K)X(S/T)) consensus sequences for PKA phosphorylation as well as several monobasic and low affinity sites, most of them located in the R domain (1Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5811) Google Scholar). Although the overall sequence identity of CFTR R domains is low, the phosphorylation sites are remarkably conserved among species (5Dahan D. Evagelidis A. Hanrahan J.W. Hinkson D.A. Jia Y. Luo J. Zhu T. Pflugers Arch. 2001; 443: S92-S96Crossref PubMed Scopus (59) Google Scholar). It is believed that the R domain combines both inhibitory and stimulatory effects. Effectively, deletion of the residues 708-835 from the R domain (6Rich D.P. Gregory R.J. Anderson M.P. Manavalan P. Smith A.E. Welsh M.J. Science. 1991; 253: 205-207Crossref PubMed Scopus (193) Google Scholar, 7Ma J. Zhao J. Drumm M.L. Xie J. Davis P.B. J. Biol. Chem. 1997; 272: 28133-28141Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and even of the residues 760-783 (8Baldursson O. Ostedgaard L.S. Rokhlina T. Cotten J.F. Welsh M.J. J. Biol. Chem. 2001; 276: 1904-1910Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), generates constitutively active channels. Moreover, overexpression or addition of the unphosphorylated R domain, encompassing residues 590-858, inhibits chloride transport (9Ma J. Tasch J.E. Tao T. Zhao J. Xie J. Drumm M.L. Davis P.B. J. Biol. Chem. 1996; 271: 7351-7356Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). PKA phosphorylation relieves this inhibition. On the other hand, it has been shown that exogenous phosphorylated R domain (either residues 590-858, 645-834, or 708-831) increases the open probability of a CFTR channel construct missing residues 708-835 from the R domain (7Ma J. Zhao J. Drumm M.L. Xie J. Davis P.B. J. Biol. Chem. 1997; 272: 28133-28141Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 10Winter M.C. Welsh M.J. Nature. 1997; 389: 294-296Crossref PubMed Scopus (124) Google Scholar, 11Ostedgaard L.S. Baldursson O. Vermeer D.W. Welsh M.J. Robertson A.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5657-5662Crossref PubMed Scopus (92) Google Scholar). Nevertheless, the effect of the phosphorylation sites seems quite unspecific. Effectively, it has been shown that PKA phosphorylates the R domain with a stoichiometry of 5 mol/mol in vitro. Site-directed mutagenesis and tryptic phosphopeptide mapping showed the phosphorylation of 5 serines in vivo (Ser-660, Ser-700, Ser-737, Ser-795, and Ser-813) (12Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (519) Google Scholar, 13Picciotto M.R. Cohn J.A. Bertuzzi G. Greengard P. Nairn A.C. J. Biol. Chem. 1992; 267: 12742-12752Abstract Full Text PDF PubMed Google Scholar). Mass spectrometry allows the detection of 8 phosphorylated serines (the same sites, plus Ser-712, Ser-768, and Ser-753) (14Neville D.C. Rozanas C.R. Price E.M. Gruis D.B. Verkman A.S. Townsend R.R. Protein Sci. 1997; 6: 2436-2445Crossref PubMed Scopus (234) Google Scholar, 15Townsend R.R. Lipniunas P.H. Tulk B.M. Verkman A.S. Protein Sci. 1996; 5: 1865-1873Crossref PubMed Scopus (42) Google Scholar). Nevertheless, the mutation of all these serines as well as the mutation of the 10 dibasic consensus sites, did not prevent PKA-dependent opening of the channel (16Chang X.B. Tabcharani J.A. Hou Y.X. Jensen T.J. Kartner N. Alon N. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1993; 268: 11304-11311Abstract Full Text PDF PubMed Google Scholar), suggesting that phosphorylation of monobasic and low affinity phosphorylation sites can relieve, partly at least, the inhibitory effect of the R domain. Furthermore, the substitution of serines by negatively charged aspartates relieves R domain inhibition (17Rich D.P. Berger H.A. Cheng S.H. Travis S.M. Saxena M. Smith A.E. Welsh M.J. J. Biol. Chem. 1993; 268: 20259-20267Abstract Full Text PDF PubMed Google Scholar), suggesting that the negative charges of the phosphate groups play a major role in this process. Even though the role of phosphorylation on CFTR activity has been widely studied, the only structural information regarding CFTR phosphorylation that has been obtained so far concerned soluble R domains (11Ostedgaard L.S. Baldursson O. Vermeer D.W. Welsh M.J. Robertson A.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5657-5662Crossref PubMed Scopus (92) Google Scholar, 18Dulhanty A.M. Riordan J.R. Biochemistry. 1994; 33: 4072-4079Crossref PubMed Scopus (93) Google Scholar). In the present study, we reconstituted active CFTR in proteoliposomes and analyzed the effect of PKA phosphorylation both on the entire protein and on specific regions of CFTR distinct from the R domain. 1H/2H exchange combined with polarized and unpolarized ATR-FTIR spectroscopy, as well as tryptophan fluorescence quenching by acrylamide were used to detect modifications of solvent accessibility occurring specifically in the transmembrane or cytosolic domains of CFTR upon PKA phosphorylation of the R domain. Materials—D2O was from Merck, nickel-nitrilotriacetic acid resin was from Qiagen, m3a7 CFTR primary antibody and anti-mouse secondary antibody were from Chemicon, ECL+ Western blot detection kit was from Amersham Biosciences, pentadecafluorooctanoic acid (PFO) was from Fluorochem, SM2 Bio-Beads were from Bio-Rad, bovine c-AMP-dependant protein kinase A catalytic domain was from Promega, and bovine alkaline phosphatase was from Sigma. All other reagents were from Merck and Sigma. Homemade software programs necessary for the analysis of IR spectra were written in Matlab (The Mathworks). Production and Purification of CFTR-His Proteins—Procedures describing production and purification of CFTR-His proteins were published previously (19Ramjeesingh M. Li C. Garami E. Huan L.J. Hewryk M. Wang Y. Galley K. Bear C.E. Biochem. J. 1997; 327: 17-21Crossref PubMed Scopus (43) Google Scholar, 20Ramjeesingh M. Garami E. Galley K. Li C. Wang Y. Bear C.E. Methods Enzymol. 1999; 294: 227-246Crossref PubMed Scopus (12) Google Scholar). Briefly, Sf9-baculovirus expression system was used for large scale production of wild type CFTR. Crude plasma membranes from frozen Sf9 cell pellets expressing recombinant CFTR-His proteins were solubilized in 8% PFO, 25 mm phosphate, pH 8.0. Purification of CFTR-His protein was performed using nickel affinity chromatography. The solubilized sample was applied to a freshly generated nickel column at a rate of 1 ml/min. A pH gradient of 8.0-6.0 was then applied to the column using a liquid chromatography pump and gradient former from Bio-Rad. 5-ml fractions were collected. Dot blot was used for CFTR detection in the fractions eluted from the column. Immunopositive fractions were selected and further analyzed by silver-stained 6% SDS/polyacrylamide gel electrophoresis and Western blotting. Fractions of high purity were then concentrated in a Centricon YM-100 concentrator from Millipore. Reconstitution of CFTR-His Proteins—A film of asolectin was formed by evaporation of chloroform under a stream of N2, followed by overnight drying under vacuum. Liposomes were prepared by sonication of the lipid film (7 min, on a 250-watt Vibra Cell Sonifier) in 6.5 ml of reconstitution buffer (20 mm Tris-HCl, 75 mm NaCl, 0.5 mm EDTA, and 1 mm dithiothreitol, pH 7.2). The sonicated phospholipid solution containing 1 mg of lipids was mixed with 50 μl of PFO 3%. The mixture was stirred continuously for 20 min at room temperature. Purified protein, dissolved in 4% PFO, was added to lipid/detergent-mixed micelles, at a 1/20 (w/w) protein/lipid ratio, and the volume was adjusted to 1 ml with reconstitution buffer. The detergent/protein/phospholipid mixture was stirred for 30 min at room temperature, and the detergent was removed by six incubations with SM2 Bio-Beads (previously washed with methanol and MilliQ water) at 4 °C. The supernatant collected from SM2 Bio-Beads was mixed with an equal volume of 80% sucrose and overlaid with a 30 to 10% sucrose linear gradient. After an overnight centrifugation at 120,000 × g in a Beckman L7 ultracentrifuge, the gradient was fractionated from the bottom of the tube. The phospholipids and protein distribution were measured, respectively, by the enzymatic colorimetric assay for phosphatidylcholine (Roche Applied Science) and by tryptophan fluorescence (excitation = 290 nm, and emission = 330 nm). Phosphorylation and Dephosphorylation of CFTR-His Proteins—Half of the reconstituted CFTR protein batch was phosphorylated by the catalytic subunit of PKA for 1 h at room temperature (20Ramjeesingh M. Garami E. Galley K. Li C. Wang Y. Bear C.E. Methods Enzymol. 1999; 294: 227-246Crossref PubMed Scopus (12) Google Scholar). Phosphorylation reaction mixture contained 250 nm catalytic subunit of PKA, 1 mm MgCl2, and 500 μm ATP in 50 mm Tris-HCl, 50 mm NaCl, pH 7.5. The other half of the reconstituted protein was dephosphorylated in the same reaction mixture with alkaline phosphatase in place of PKA. PKA and alkaline phosphatase were removed by ultracentrifugation at 180,000 × g for 2 h. The pellets containing the proteoliposomes were resuspended in 2 mm Hepes, 150 mm NaCl, pH 7.2, and the absence of residual kinase and phosphatase was assessed by 6% SDS/polyacrylamide gel electrophoresis. Uptake of Chloride by Proteoliposomes—A concentrative tracer uptake assay, described previously (20Ramjeesingh M. Garami E. Galley K. Li C. Wang Y. Bear C.E. Methods Enzymol. 1999; 294: 227-246Crossref PubMed Scopus (12) Google Scholar), was used to measure 36Cl- flux into proteoliposomes containing purified and reconstituted CFTR protein. Intravesicular 36Cl- was assayed after incubation of proteoliposomes at 33 °C for 60 min, in the presence of MgATP, for phosphorylated and dephosphorylated samples. Attenuated Total Reflection Fourier Transform IR Spectroscopy— ATR-FTIR spectra were recorded, at room temperature, on a Bruker IFS55 FTIR spectrophotometer equipped with a liquid nitrogen-cooled mercury-cadmium-telluride detector at a nominal resolution of 2 cm-1 and encoded every 1 cm-1. The spectrophotometer was continuously purged with air dried on a FTIR purge gas generator (75-62 Balston, Maidstone, UK). The internal reflection element (ATR) was a germanium plate (50 × 20 × 2 mm) with an aperture angle of 45°, yielding 25 internal reflections (21Goormaghtigh E. Raussens V. Ruysschaert J.M. Biochim. Biophys. Acta. 1999; 1422: 105-185Crossref PubMed Scopus (499) Google Scholar). Sample Preparation—Thin films of oriented multilayers were obtained by slowly evaporating the sample on one side of the ATR plate under a stream of nitrogen (22Fringeli U.P. Gunthard H.H. Mol. Biol. Biochem. Biophys. 1981; 31: 270-332Crossref PubMed Scopus (311) Google Scholar). The ATR plate was then sealed in a universal sample holder. The sample contained 20 μg of reconstituted phosphorylated or dephosphorylated CFTR. Secondary Structure Analysis—512 scans were averaged for each measurement. The spectra were corrected for atmospheric water absorption as described previously (23Goormaghtigh E. de Jongh H.H. Ruysschaert J.M. Appl. Spectrosc. 1996; 50: 1519-1527Crossref Scopus (43) Google Scholar, 24Grimard V. Vigano C. Margolles A. Wattiez R. van Veen H.W. Konings W.N. Ruysschaert J.M. Goormaghtigh E. Biochemistry. 2001; 40: 11876-11886Crossref PubMed Scopus (35) Google Scholar). Secondary structure content was determined by comparison with the spectra of 50 proteins from a reference data base. For all spectra, a baseline was subtracted between 1700, 1600, and 1500 cm-1, and the spectra were normalized between 1700 and 1500 cm-1. A linear model containing two wavenumbers was constructed from the reference data base for each secondary structure and applied to the CFTR spectra. Both the protein data base and the mathematical model have been described previously in the case of circular dichroism (25Raussens V. Ruysschaert J.M. Goormaghtigh E. Anal. Biochem. 2003; 319: 114-121Crossref PubMed Scopus (109) Google Scholar). Kinetics of Deuteration—Films containing 20 μg of reconstituted phosphorylated or dephosphorylated CFTR were prepared on a germanium plate as described above. Before starting the deuteration, 10 spectra of the sample were recorded to test the stability of the measurements and the reproducibility of the area determination. At zero time, a 2H2O-saturated N2 flux, at a flow rate of 100 ml/min (controlled by a Brooks flow meter), was applied to the sample. For each kinetic time point, 24 spectra were recorded and averaged at a resolution of 2 cm-1. The resulting spectra were corrected for atmospheric water absorption and side-chain contribution as described previously (23Goormaghtigh E. de Jongh H.H. Ruysschaert J.M. Appl. Spectrosc. 1996; 50: 1519-1527Crossref Scopus (43) Google Scholar, 24Grimard V. Vigano C. Margolles A. Wattiez R. van Veen H.W. Konings W.N. Ruysschaert J.M. Goormaghtigh E. Biochemistry. 2001; 40: 11876-11886Crossref PubMed Scopus (35) Google Scholar). The area of Amide II, characteristic of the δ(N-H) vibration, was obtained by integration between 1575 and 1505 cm-1. For each spectrum, the area of Amide II was divided by the area of the lipid ester vibration band integrated between 1780 and 1700 cm-1, to correct for any change in total intensity of the spectra during the deuteration process. This ratio, expressed as a percentage was plotted versus deuteration time. The 100% value is defined by the Amide II/lipid ratio obtained before deuteration. The 0% value corresponds to a zero absorption in the Amide II region, observed for the full deuteration of a protein (27Goormaghtigh E. Cabiaux V. Ruysschaert J.M. Subcell. Biochem. 1994; 23: 363-403Crossref PubMed Scopus (138) Google Scholar). Polarized ATR-FTIR Spectroscopy—Films containing 20 μg of reconstituted phosphorylated or dephosphorylated CFTR were prepared on a germanium plate as described above. Spectra were recorded with the incident light polarized parallel and perpendicular with respect to the incidence plane before and after 12-h deuteration. For each spectrum, 512 scans were recorded and averaged, and the resulting spectra were corrected for atmospheric water absorption (23Goormaghtigh E. de Jongh H.H. Ruysschaert J.M. Appl. Spectrosc. 1996; 50: 1519-1527Crossref Scopus (43) Google Scholar, 24Grimard V. Vigano C. Margolles A. Wattiez R. van Veen H.W. Konings W.N. Ruysschaert J.M. Goormaghtigh E. Biochemistry. 2001; 40: 11876-11886Crossref PubMed Scopus (35) Google Scholar). Dichroism spectra were computed by subtracting the perpendicular polarized spectrum from the parallel polarized spectrum. The subtraction coefficient was chosen so that the area of the lipid ester band equals zero on the dichroism spectrum, to take into account the difference in the relative power of the evanescent field for each polarization as described before (29Bechinger B. Ruysschaert J.M. Goormaghtigh E. Biophys. J. 1999; 76: 552-563Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Fluorescence Quenching Experiments—Acrylamide quenching experiments were carried out on a SLM Aminco 8000 fluorometer at an excitation wavelength of 290 nm to reduce the absorbance of acrylamide. Acrylamide aliquots were added from a 3 m solution to the proteoliposome suspension containing 5 μg of reconstituted phosphorylated or dephosphorylated CFTR. Fluorescence intensities were measured at 330 nm after each addition of quencher. All measurements were carried out at 25 °C. Acrylamide quenching data were analyzed according to the Stern-Volmer equation for collisional quenching (30Lehrer S.S. Biochemistry. 1971; 10: 3254-3263Crossref PubMed Scopus (1654) Google Scholar). Purification and Active Reconstitution of CFTR—The use of biophysical techniques such as ATR-FTIR and fluorescence spectroscopy requires purified and reconstituted proteoliposomes with a relatively high protein to lipid ratio (21Goormaghtigh E. Raussens V. Ruysschaert J.M. Biochim. Biophys. Acta. 1999; 1422: 105-185Crossref PubMed Scopus (499) Google Scholar, 30Lehrer S.S. Biochemistry. 1971; 10: 3254-3263Crossref PubMed Scopus (1654) Google Scholar). Purification of His-tagged CFTR was performed as previously described (19Ramjeesingh M. Li C. Garami E. Huan L.J. Hewryk M. Wang Y. Galley K. Bear C.E. Biochem. J. 1997; 327: 17-21Crossref PubMed Scopus (43) Google Scholar) and yielded a well purified protein as shown on a silver-stained polyacrylamide gel and by Western blot (see Fig. 1). The diffuse band is due to the variation of glycosylation considering the expression in Sf9 insect cells (31Kartner N. Hanrahan J.W. Jensen T.J. Naismith A.L. Sun S.Z. Ackerley A.A. Reyes E.F. Tsui L.C. Rommens J.M. Bear C.E. Riordan J.R. Cell. 1991; 64: 681-691Abstract Full Text PDF PubMed Scopus (391) Google Scholar). To reconstitute CFTR, Bio-Beads were used to remove the detergent (32Rigaud J.L. Pitard B. Levy D. Biochim. Biophys. Acta. 1995; 1231: 223-246Crossref PubMed Scopus (393) Google Scholar). After several incubations with the Bio-Beads, the sample was applied to a sucrose gradient (Fig. 2). A homogeneous population of proteoliposomes migrated at a density of 1.10 g/cm3. No protein was observed elsewhere in the gradient. The diameter of these proteoliposomes evaluated by photon correlation spectroscopy was found to be 148.0 ± 0.8 nm (results not shown). These proteoliposomes appeared to be suitable for spectroscopic purposes, with a protein to lipid ratio of 1/30 (w/w).Fig. 2Sucrose gradient profile of actively reconstituted CFTR. After centrifugation, fractions were collected from the bottom to the top of the gradient and measured for protein (solid line) and lipid (dashed line) content as described under “Experimental Procedures.” The y-axis represents the percentage of proteins or lipids in a fraction relative to the total amount.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Extensive phosphorylation by PKA or dephosphorylation by alkaline phosphatase was performed as previously described (20Ramjeesingh M. Garami E. Galley K. Li C. Wang Y. Bear C.E. Methods Enzymol. 1999; 294: 227-246Crossref PubMed Scopus (12) Google Scholar). The chloride channel activity of this sample was evaluated by monitoring the uptake of radioactive chloride ions. Fig. 3 shows that phosphorylated CFTR was effectively able to transport chloride ions at a rate of 145 nmol/μg of protein per hour. When CFTR was dephosphorylated, the chloride flux drops significantly to a rate of 80 nmol/μg of protein. Phosphorylation-induced Conformational Changes—The reconstituted sample was used for ATR-FTIR experiments. The IR spectrum of a protein is mainly characterized by two bands, the Amide I and Amide II, due mainly to the absorption of the amide ν(C=O) and δ(N-H) vibrations, respectively (21Goormaghtigh E. Raussens V. Ruysschaert J.M. Biochim. Biophys. Acta. 1999; 1422: 105-185Crossref PubMed Scopus (499) Google Scholar). The Amide I is located between 1700 and 1600 cm-1, and its shape is particularly sensitive to the secondary structure (33Goormaghtigh E. Cabiaux V. Ruysschaert J.M. Subcell. Biochem. 1994; 23: 405-450Crossref PubMed Scopus (351) Google Scholar). The comparison of the Amide I shape of phosphorylated and dephosphorylated CFTR shown in Fig. 4 reveals no significant change. Secondary structure content was further determined for the two samples (Table I) by comparison with the spectra of a 50-protein data base (25Raussens V. Ruysschaert J.M. Goormaghtigh E. Anal. Biochem. 2003; 319: 114-121Crossref PubMed Scopus (109) Google Scholar). Due to the proximity of β-strands and random coil absorption in the Amide 1 band (33Goormaghtigh E. Cabiaux V. Ruysschaert J.M. Subcell. Biochem. 1994; 23: 405-450Crossref PubMed Scopus (351) Google Scholar), a small underestimation of the β-strands content is possible. However, we can see that no significant change of secondary structure occurs upon CFTR phosphorylation.Table ISecondary structure of CFTR Secondary structures were determined as described under “Experimental Procedures” for dephosphorylated (CFTR-dP) and phosphorylated (CFTR-P) CFTR. Numbers are given in percentages. Values are the mean of three experiments and are given with indication of the standard deviation.α-Helicesβ-Strandsβ-TurnsRandom coilCFTR-dP45 ± 41 ± 117 ± 237 ± 3CFTR-P47 ± 22 ± 216 ± 235 ± 2 Open table in a new tab Because the tertiary structure could change in the absence of significant secondary structure modification, we monitored 1H/2H exchange kinetics for the phosphorylated and the dephosphorylated samples. After exposure to 2H2O, the labile hydrogens from the amide bonds are exchanged by deuterium. This results in the decrease of the area of the Amide II band located between 1575 and 1505 cm-1. This decrease can be related to the solvent accessibility and the secondary structures stability (Fig. 5) (27Goormaghtigh E. Cabiaux V. Ruysschaert J.M. Subcell. Biochem. 1994; 23: 363-403Crossref PubMed Scopus (138) Google Scholar). The exchange observed here is fast in comparison with other proteins, because 70% of the amide hydrogens are already exchanged after 20 min. The phosphorylation induces an increase of the 1H/2H exchange, giving rise to 90% of exchange in the same time interval (Fig. 6). These exchange curves can be fitted by a multiexponential decay corresponding to the different groups of amide protons exchanging at various rates (27Goormaghtigh E. Cabiaux V. Ruysschaert J.M. Subcell. Biochem. 1994; 23: 363-403Crossref PubMed Scopus (138) Google Scholar). Table II shows that the population of the fast exchanging protons is enhanced by 32% upon phosphorylation. A corresponding decrease is observed in the two others proton populations.Fig. 6Exchange curves for actively reconstituted CFTR. Evolution of the proportion of unexchanged amide bonds (H(t)) computed between 0 and 100% presented as a function of the deuteration time for phosphorylated (×) and dephosphorylated (○) CFTR. Only the first 50 min are shown. Error bars represent the standard deviation computed for four independent experiments. The solid lines were obtained by a fitting procedure of exponential decay.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIProportion (a1, a2, a3) of the three exponential components characterized by a half-decay of T1 = 0.6, T2 = 10, and T3 = 2000 min Exchange curves were fitted by an exponential decay of equation, H(t) = Σai*exp(-t/Ti), for dephosphorylated (DP) and phosphorylated (P) CFTR.Phosphorylation stateNPPa138% (562)aNumbers in parentheses correspond to the number of amino acids involved70% (1036)a235% (518)21% (311)a327% (400)9% (133)a Numbers in parentheses correspond to the number of amino acids involved Open table in a new tab Location of the Phosphorylation-induced Conformational Changes—To locate more specifically the conformational changes induced by phosphorylation, two approaches were used to detect structural changes occurring specifically in the cytosolic and membrane domains, respectively, upon phosphorylation of the R domain. The first one takes advantage of the intrinsic fluorescence of tryptophan residues. Tryptophan residues give rise to a fluorescence emission with a maximum wavelength at 330 nm. The aqueous quencher acrylamide induces a decrease of the fluorescence, which depends on the accessibility of the cytosolic tryptophans to the quencher (34Eftink M.R. Ghiron C.A. Biochemistry. 1976; 15: 672-680Crossref PubMed Scopus (986) Google Scholar). Because no tryptophans are located in the R domain, the measure will reflect changes occurring outside this domain. Quenching was observed for the phosphorylated as well as for the dephosphorylated samples, but the intensity of the effect was more pronounced for the phosphorylated CFTR, demonstrating that the tryptophans are more accessible to acrylamide in this conformational state (Fig. 7). The second method was developed previously in our laboratory (24Grimard V. Vigano C. Margolles A. Wattiez R. van Veen H.W. Konings W.N. Ruysschaert J.M. Goormaghtigh E. Biochemistry. 2001; 40: 11876-11886Crossref PubMed Scopus (35) Google Scholar, 36Vigano C. Grimard V. Margolles A. Goormaghtigh E. van Veen H.W. Konings W.N. Ruysschaert J.M. FEBS Lett. 2002; 530: 197-203Crossref PubMed Scopus (13) Google Scholar). It is based on the use of polarized" @default.
• W2050179344 created "2016-06-24" @default.
• W2050179344 creator A5002189140 @default.
• W2050179344 creator A5003858064 @default.
• W2050179344 creator A5023233738 @default.
• W2050179344 creator A5033878654 @default.
• W2050179344 creator A5068990419 @default.
• W2050179344 creator A5085975282 @default.
• W2050179344 date "2004-02-01" @default.
• W2050179344 modified "2023-09-26" @default.
• W2050179344 title "Phosphorylation-induced Conformational Changes of Cystic Fibrosis Transmembrane Conductance Regulator Monitored by Attenuated Total Reflection-Fourier Transform IR Spectroscopy and Fluorescence Spectroscopy" @default.
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