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• W2054523600 abstract "CP12 is a protein of 8.7 kDa that contributes to Calvin cycle regulation by acting as a scaffold element in the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in photosynthetic organisms. NMR studies of recombinant CP12 (isoform 2) of Arabidopsis thaliana show that CP12-2 is poorly structured. CP12-2 is monomeric in solution and contains four cysteines, which can form two intramolecular disulfides with midpoint redox potentials of –326 and –352 mV, respectively, at pH 7.9. Site-specific mutants indicate that the C-terminal disulfide is involved in the interaction between CP12-2 and GAPDH (isoform A4), whereas the N-terminal disulfide is involved in the interaction between this binary complex and PRK. In the presence of NAD, oxidized CP12-2 interacts with A4-GAPDH (KD = 0.18 μm) to form a binary complex of 170 kDa with (A4-GAPDH)-(CP12-2)2 stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering analysis. PRK is a dimer and by interacting with this binary complex (KD = 0.17 μm) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A4-GAPDH)-(CP12-2)2-(PRK))2. Thermodynamic parameters indicate that assembly of both binary and ternary complexes is exoergonic although penalized by a decrease in entropy that suggests an induced folding of CP12-2 upon binding to partner proteins. The redox dependence of events leading to supramolecular complexes is consistent with a role of CP12 in coordinating the reversible inactivation of chloroplast enzymes A4-GAPDH and PRK during darkness in photosynthetic tissues. CP12 is a protein of 8.7 kDa that contributes to Calvin cycle regulation by acting as a scaffold element in the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in photosynthetic organisms. NMR studies of recombinant CP12 (isoform 2) of Arabidopsis thaliana show that CP12-2 is poorly structured. CP12-2 is monomeric in solution and contains four cysteines, which can form two intramolecular disulfides with midpoint redox potentials of –326 and –352 mV, respectively, at pH 7.9. Site-specific mutants indicate that the C-terminal disulfide is involved in the interaction between CP12-2 and GAPDH (isoform A4), whereas the N-terminal disulfide is involved in the interaction between this binary complex and PRK. In the presence of NAD, oxidized CP12-2 interacts with A4-GAPDH (KD = 0.18 μm) to form a binary complex of 170 kDa with (A4-GAPDH)-(CP12-2)2 stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering analysis. PRK is a dimer and by interacting with this binary complex (KD = 0.17 μm) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A4-GAPDH)-(CP12-2)2-(PRK))2. Thermodynamic parameters indicate that assembly of both binary and ternary complexes is exoergonic although penalized by a decrease in entropy that suggests an induced folding of CP12-2 upon binding to partner proteins. The redox dependence of events leading to supramolecular complexes is consistent with a role of CP12 in coordinating the reversible inactivation of chloroplast enzymes A4-GAPDH and PRK during darkness in photosynthetic tissues. The photosynthetic reduction cycle for carbon organication (Calvin cycle) is a finely regulated metabolism that plants keep tuned with light reactions of photosynthesis under variable environmental conditions. Thioredoxins and metabolic intermediates play essential signaling roles within this complex regulatory system (1Wolosiuk R.A. Ballicora M.A. Hagelin K. FASEB J. 1993; 7: 622-637Crossref PubMed Scopus (59) Google Scholar). Two nonconsecutive enzymes of the Calvin cycle, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 2The abbreviations used are: GAPDHglyceraldehyde-3-phosphate dehydrogenasePRKphosphoribulokinaseITCisothermal titration calorimetryMALSmultiangle light scatteringQELSquasielastic light scatteringCTEC-terminal extension of GAPDH subunit BDTTdithiothreitolDTNB5,5′-dithiobis-(2-nitrobenzoic acid). 2The abbreviations used are: GAPDHglyceraldehyde-3-phosphate dehydrogenasePRKphosphoribulokinaseITCisothermal titration calorimetryMALSmultiangle light scatteringQELSquasielastic light scatteringCTEC-terminal extension of GAPDH subunit BDTTdithiothreitolDTNB5,5′-dithiobis-(2-nitrobenzoic acid). and phosphoribulokinase (PRK), are regulated in this way, both individually and through the reversible formation of supramolecular complexes (2Wedel N. Soll J. Paap B.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10479-10484Crossref PubMed Scopus (128) Google Scholar, 3Wedel N. Soll J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9699-9704Crossref PubMed Scopus (138) Google Scholar, 4Scheibe R. Wedel N. Vetter S. Emmerlich V. Sauermann S.M. Eur. J. Biochem. 2002; 269: 5617-5624Crossref PubMed Scopus (68) Google Scholar, 5Graciet E. Lebreton S. Gontero B. J. Exp. Bot. 2004; 55: 1245-1254Crossref PubMed Scopus (93) Google Scholar, 6Tamoi M. Miyazaki T. Fukamizo T. Shigeoka S. Plant J. 2005; 42: 504-513Crossref PubMed Scopus (165) Google Scholar, 7Marri L. Trost P. Pupillo P. Sparla F. Plant Physiol. 2005; 139: 1433-1443Crossref PubMed Scopus (65) Google Scholar, 8Trost P. Fermani S. Marri L. Zaffagnini M. Falini G. Scagliarini S. Pupillo P. Sparla F. Photosynth. Res. 2006; 89: 263-275Crossref PubMed Scopus (73) Google Scholar).Chloroplast GAPDH is mainly heteromeric in land plants, with homologous A and B subunits occurring in stoichiometric ratio (9Cerff R. Chambers S.E. J. Biol. Chem. 1979; 254: 6094-6098Abstract Full Text PDF PubMed Google Scholar, 10Fermani S. Sparla F. Falini G. Martelli P.L. Casadio R. Pupillo P. Ripamonti A. Trost P. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11109-11114Crossref PubMed Scopus (64) Google Scholar). The B-subunits confer regulatory properties, and the enzyme oscillates between a fully active A2B2 tetramer at one extreme and a partially inhibited A8B8 hexadecamer at the other (11Pupillo P. Giuliani Piccari G. Eur. J. Biochem. 1975; 51: 475-482Crossref PubMed Scopus (74) Google Scholar). Partially polymerized intermediates like A4B4 have also been reported (12Trost P. Scagliarini S. Valenti V. Pupillo P. Planta. 1993; 190: 320-326Crossref Scopus (36) Google Scholar, 13Baalmann E. Backhausen J.E. Kitzmann C. Scheibe R. Bot. Acta. 1994; 107: 313-320Crossref Scopus (44) Google Scholar, 14Scagliarini S. Trost P. Pupillo P. J. Exp. Bot. 1998; 49: 1307-1315Crossref Scopus (28) Google Scholar). Thioredoxins and metabolites directly regulate AB-GAPDH activity and strongly affect the equilibrium between active tetramers and aggregated forms (8Trost P. Fermani S. Marri L. Zaffagnini M. Falini G. Scagliarini S. Pupillo P. Sparla F. Photosynth. Res. 2006; 89: 263-275Crossref PubMed Scopus (73) Google Scholar, 10Fermani S. Sparla F. Falini G. Martelli P.L. Casadio R. Pupillo P. Ripamonti A. Trost P. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11109-11114Crossref PubMed Scopus (64) Google Scholar).A second isoform of chloroplast GAPDH in land plants is a stable homotetramer of A-subunits (A4-GAPDH) (9Cerff R. Chambers S.E. J. Biol. Chem. 1979; 254: 6094-6098Abstract Full Text PDF PubMed Google Scholar, 15Fermani S. Ripamonti A. Sabatino P. Zanotti G. Scagliarini S. Sparla F. Trost P. Pupillo P. J. Mol. Biol. 2001; 314: 527-542Crossref PubMed Scopus (35) Google Scholar), similar to Calvin cycle GAPDH of lower photosynthetic organisms (5Graciet E. Lebreton S. Gontero B. J. Exp. Bot. 2004; 55: 1245-1254Crossref PubMed Scopus (93) Google Scholar, 6Tamoi M. Miyazaki T. Fukamizo T. Shigeoka S. Plant J. 2005; 42: 504-513Crossref PubMed Scopus (165) Google Scholar, 16Figge R.M. Schubert M. Brinkmann H. Cerff R. Mol. Biol. Evol. 1999; 16: 429-440Crossref PubMed Scopus (89) Google Scholar). A4-GAPDH only accounts for a minor portion of total chloroplast GAPDH activity in land plants (14Scagliarini S. Trost P. Pupillo P. J. Exp. Bot. 1998; 49: 1307-1315Crossref Scopus (28) Google Scholar, 15Fermani S. Ripamonti A. Sabatino P. Zanotti G. Scagliarini S. Sparla F. Trost P. Pupillo P. J. Mol. Biol. 2001; 314: 527-542Crossref PubMed Scopus (35) Google Scholar). Due to the absence of B-subunits, A4-GAPDH is not directly regulated by thioredoxins and metabolites (8Trost P. Fermani S. Marri L. Zaffagnini M. Falini G. Scagliarini S. Pupillo P. Sparla F. Photosynth. Res. 2006; 89: 263-275Crossref PubMed Scopus (73) Google Scholar, 17Baalmann E. Scheibe R. Cerff R. Martin W. Plant Mol. Biol. 1996; 32: 505-513Crossref PubMed Scopus (63) Google Scholar, 18Li A.D. Anderson L.E. Plant Physiol. 1997; 115: 1201-1209Crossref PubMed Scopus (32) Google Scholar), although the reversible glutathionylation of the active site cysteine 149 provides a mechanism of A4-GAPDH regulation that may be relevant under stress (19Zaffagnini M. Michelet L. Marchand C. Sparla F. Decottignies P. Le Maréchal P. Miginiac-Maslow M. Noctor G. Trost P. Lemaire S.D. FEBS J. 2007; 274: 212-226Crossref PubMed Scopus (102) Google Scholar). Alternatively, reversible down-regulation of A4-GAPDH activity can be achieved through formation of a supramolecular complex with PRK and the regulatory peptide CP12 (2Wedel N. Soll J. Paap B.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10479-10484Crossref PubMed Scopus (128) Google Scholar, 3Wedel N. Soll J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9699-9704Crossref PubMed Scopus (138) Google Scholar, 4Scheibe R. Wedel N. Vetter S. Emmerlich V. Sauermann S.M. Eur. J. Biochem. 2002; 269: 5617-5624Crossref PubMed Scopus (68) Google Scholar, 5Graciet E. Lebreton S. Gontero B. J. Exp. Bot. 2004; 55: 1245-1254Crossref PubMed Scopus (93) Google Scholar, 6Tamoi M. Miyazaki T. Fukamizo T. Shigeoka S. Plant J. 2005; 42: 504-513Crossref PubMed Scopus (165) Google Scholar, 7Marri L. Trost P. Pupillo P. Sparla F. Plant Physiol. 2005; 139: 1433-1443Crossref PubMed Scopus (65) Google Scholar, 8Trost P. Fermani S. Marri L. Zaffagnini M. Falini G. Scagliarini S. Pupillo P. Sparla F. Photosynth. Res. 2006; 89: 263-275Crossref PubMed Scopus (73) Google Scholar, 20Graciet E. Gans P. Wedel N. Lebreton S. Camadro J.M. Gontero B. Biochemistry. 2003; 42: 8163-8170Crossref PubMed Scopus (98) Google Scholar). In land plants, PRK itself undergoes a light/dark modulation mediated by thioredoxins (21Porter M.A. Stringer C.D. Hartman F.C. J. Biol. Chem. 1988; 263: 123-129Abstract Full Text PDF PubMed Google Scholar), but once incorporated into the complex, both A4-GAPDH and PRK activities are inhibited in a coordinated manner (7Marri L. Trost P. Pupillo P. Sparla F. Plant Physiol. 2005; 139: 1433-1443Crossref PubMed Scopus (65) Google Scholar). Similar to A8B8-GAPDH, the stability of the supramolecular complex involving A4-GAPDH, CP12, and PRK is controlled by thioredoxins and several cofactors, including NAD(H) and NADP(H), ATP, and 1,3-bisphosphoglycerate (3Wedel N. Soll J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9699-9704Crossref PubMed Scopus (138) Google Scholar, 4Scheibe R. Wedel N. Vetter S. Emmerlich V. Sauermann S.M. Eur. J. Biochem. 2002; 269: 5617-5624Crossref PubMed Scopus (68) Google Scholar, 5Graciet E. Lebreton S. Gontero B. J. Exp. Bot. 2004; 55: 1245-1254Crossref PubMed Scopus (93) Google Scholar, 6Tamoi M. Miyazaki T. Fukamizo T. Shigeoka S. Plant J. 2005; 42: 504-513Crossref PubMed Scopus (165) Google Scholar, 7Marri L. Trost P. Pupillo P. Sparla F. Plant Physiol. 2005; 139: 1433-1443Crossref PubMed Scopus (65) Google Scholar). It is generally agreed that aggregated forms of GAPDH (A8B8) and GAPDH-CP12-PRK complexes are prevalent in chloroplasts in the dark, whereas illumination favors the accumulation of fully active GAPDH tetramers (A2B2, A4) and PRK dimers (4Scheibe R. Wedel N. Vetter S. Emmerlich V. Sauermann S.M. Eur. J. Biochem. 2002; 269: 5617-5624Crossref PubMed Scopus (68) Google Scholar, 5Graciet E. Lebreton S. Gontero B. J. Exp. Bot. 2004; 55: 1245-1254Crossref PubMed Scopus (93) Google Scholar, 6Tamoi M. Miyazaki T. Fukamizo T. Shigeoka S. Plant J. 2005; 42: 504-513Crossref PubMed Scopus (165) Google Scholar, 8Trost P. Fermani S. Marri L. Zaffagnini M. Falini G. Scagliarini S. Pupillo P. Sparla F. Photosynth. Res. 2006; 89: 263-275Crossref PubMed Scopus (73) Google Scholar, 12Trost P. Scagliarini S. Valenti V. Pupillo P. Planta. 1993; 190: 320-326Crossref Scopus (36) Google Scholar, 13Baalmann E. Backhausen J.E. Kitzmann C. Scheibe R. Bot. Acta. 1994; 107: 313-320Crossref Scopus (44) Google Scholar, 22Porter M.A. Planta. 1990; 181: 349-357Crossref PubMed Scopus (13) Google Scholar, 23Baalmann E. Backhausen J.E. Rak C. Vetter S. Scheibe R. Arch. Biochem. Biophys. 1995; 324: 201-208Crossref PubMed Scopus (75) Google Scholar).The acronym CP12 refers to small proteins of nearly 80 amino acids, widespread in oxygenic photosynthetic organisms (24Pohlmeyer K. Paap B.K. Soll J. Wedel N. Plant Mol. Biol. 1996; 32: 969-978Crossref PubMed Scopus (68) Google Scholar, 25Petersen J. Teich R. Becker B. Cerff R. Brinkmann H. Mol. Biol. Evol. 2006; 23: 1109-1118Crossref PubMed Scopus (66) Google Scholar), apparently lacking an ordered structure in solution (20Graciet E. Gans P. Wedel N. Lebreton S. Camadro J.M. Gontero B. Biochemistry. 2003; 42: 8163-8170Crossref PubMed Scopus (98) Google Scholar). Interestingly, the C-terminal half of CP12 is closely related to the C-terminal extension (CTE) of GAPDH B-subunits (24Pohlmeyer K. Paap B.K. Soll J. Wedel N. Plant Mol. Biol. 1996; 32: 969-978Crossref PubMed Scopus (68) Google Scholar). Both the CTE and the C-terminal part of CP12 contain a couple of conserved cysteines, which can form an intramolecular disulfide and are potential targets of thioredoxin regulation (3Wedel N. Soll J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9699-9704Crossref PubMed Scopus (138) Google Scholar, 8Trost P. Fermani S. Marri L. Zaffagnini M. Falini G. Scagliarini S. Pupillo P. Sparla F. Photosynth. Res. 2006; 89: 263-275Crossref PubMed Scopus (73) Google Scholar, 10Fermani S. Sparla F. Falini G. Martelli P.L. Casadio R. Pupillo P. Ripamonti A. Trost P. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11109-11114Crossref PubMed Scopus (64) Google Scholar, 17Baalmann E. Scheibe R. Cerff R. Martin W. Plant Mol. Biol. 1996; 32: 505-513Crossref PubMed Scopus (63) Google Scholar, 26Sparla F. Pupillo P. Trost P. J. Biol. Chem. 2002; 277: 44946-44952Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 27Qi J. Isupov M.N. Littlechild J.A. Anderson L.E. J. Biol. Chem. 2001; 276: 35247-35252Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 28Lebreton S. Andreescu S. Graciet E. Gontero B. FEBS J. 2006; 273: 3358-3369Crossref PubMed Scopus (35) Google Scholar). In addition, most CP12 proteins contain a second pair of conserved cysteines in the N-terminal half of the molecule also able to form a disulfide bond (2Wedel N. Soll J. Paap B.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10479-10484Crossref PubMed Scopus (128) Google Scholar, 25Petersen J. Teich R. Becker B. Cerff R. Brinkmann H. Mol. Biol. Evol. 2006; 23: 1109-1118Crossref PubMed Scopus (66) Google Scholar). However, the redox properties of CP12 disulfide bonds are unknown, and it is difficult to predict whether CP12 could exist in different redox states in vivo. Studies on the green algae Chlamydomonas reinhardtii (20Graciet E. Gans P. Wedel N. Lebreton S. Camadro J.M. Gontero B. Biochemistry. 2003; 42: 8163-8170Crossref PubMed Scopus (98) Google Scholar) and the flowering plant Arabidopsis thaliana (7Marri L. Trost P. Pupillo P. Sparla F. Plant Physiol. 2005; 139: 1433-1443Crossref PubMed Scopus (65) Google Scholar) help to trace the sequence of events during the formation of CP12-mediated supramolecular complexes. First, A4-GAPDH (in complex with NAD) interacts with oxidized CP12, and then oxidized PRK can participate in the assembly of the ternary complex, resulting in strongly inhibited enzyme activities.In this paper, we shall investigate some biochemical-molecular features of A. thaliana CP12-2 and properties of the supramolecular complexes it forms with A4-GAPDH and PRK. The CP12-2 isoform, produced by one of three CP12 genes known for this species (8Trost P. Fermani S. Marri L. Zaffagnini M. Falini G. Scagliarini S. Pupillo P. Sparla F. Photosynth. Res. 2006; 89: 263-275Crossref PubMed Scopus (73) Google Scholar), was chosen as a model, since the expression pattern of the CP12-2 gene in different Arabidopsis organs and conditions strictly followed that of GAPDH, PRK, and other Calvin cycle genes (8Trost P. Fermani S. Marri L. Zaffagnini M. Falini G. Scagliarini S. Pupillo P. Sparla F. Photosynth. Res. 2006; 89: 263-275Crossref PubMed Scopus (73) Google Scholar, 29Marri L. Sparla F. Pupillo P. Trost P. J. Exp. Bot. 2005; 56: 73-80PubMed Google Scholar). The resulting model supports the view that these supramolecular complexes represent an instrument for photosynthetic organisms to finely modulate Calvin cycle turnover in response, for example, to changes in light intensity as commonly occur in natural environments and to safely and reversibly store photosynthetic enzymes in an inactive conformation during the night.EXPERIMENTAL PROCEDURESProtein Expression and Purification—Heterologous expression and purification of recombinant A4-GAPDH (At3g26650), PRK (At1g32060), CP12-2 (At3g62410), and CP12-2 site-specific mutants of A. thaliana were performed as described (7Marri L. Trost P. Pupillo P. Sparla F. Plant Physiol. 2005; 139: 1433-1443Crossref PubMed Scopus (65) Google Scholar). NMR analyses were performed on uniformly 15N-labeled His-tagged CP12-2 samples obtained by transformed Escherichia coli BL21(DE3) cells grown in M9 minimal medium containing 1 g/liter 15NH4Cl (Euriso-top, France) as the sole nitrogen source. An overnight culture of 25 ml in M9 medium was transferred to 500 ml of fresh M9 medium, both supplied with kanamycin (50 μg/ml) and grown at 37 °C under shaking. When optical density at 600 nm reached 0.6–1.0 units, expression was induced by the addition of 0.4 mm isopropyl-β-d-thiogalactopyranoside. Since M9 minimal medium prevented rapid cell growth, the induction phase was prolonged for 15 h before cells were collected by centrifugation (10,000 rpm; 15 min). CP12-2 was purified from the resulting pellet as previously described (7Marri L. Trost P. Pupillo P. Sparla F. Plant Physiol. 2005; 139: 1433-1443Crossref PubMed Scopus (65) Google Scholar). Purified proteins were quantified by absorbance at 280 nm (7Marri L. Trost P. Pupillo P. Sparla F. Plant Physiol. 2005; 139: 1433-1443Crossref PubMed Scopus (65) Google Scholar), desalted in appropriate buffers, and stored at –20 °C. Concentrations of purified proteins are all referred to native conformations (CP12-2 monomers, A4-GAPDH tetramers, and PRK dimers).CP12-2 Site-specific Mutants—Site-specific mutants of recombinant CP12-2 were obtained as previously described (26Sparla F. Pupillo P. Trost P. J. Biol. Chem. 2002; 277: 44946-44952Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). PCR primers were the following (mutation sites are underlined): C22S(up), 5′-AAGCTCAGGAGACTTCTGCGGGCGATCC-3′; C22S(down), 5′-ATCGCCCGCAGAAGTCTCCTGAGCTTCC-3′; C73S(up), 5′-ACAATCCTGAGACCAACGAGTCCCGTACTTACG-3′; C73S(down), 5′-TTGTCGTAAGTACGGGACTCGTTGGTCTCAGG-3′. The presence of mutations was confirmed by DNA sequence analysis.NMR Spectra—Uniformly 15N-labeled CP12-2, provided with His tag, was oxidized by the addition of 20 mm oxidized DTT (Sigma). After 16–18 h of incubation at 4 °C, the sample was desalted in 25 mm potassium phosphate buffer, pH 7.0, and concentrated. NMR samples were typically 300 or 600 μl of 1 mm CP12-2 solution in 25 mm potassium phosphate buffer, pH 7.0, 5% (v/v) 2H2O, 0.05% (w/v) sodium azide.Two-dimensional 1H-15N HSQC (30Bodenhausen G. Ruben D.J. Chem. Phys. Lett. 1980; 69: 185-189Crossref Scopus (2412) Google Scholar) spectra were recorded with 256 and 2048 complex points in F1 and F2 dimensions, respectively, at 20 °C on a Bruker AvanceII 800-MHz spectrometer equipped with a triple-resonance (1H, 13C, 15N) probe, including field xyz gradients.Spectra were processed using Topspin™ version 1.3 (Bruker). Chemical shifts were referenced to internal d4-3-(trimethylsily) propionic acid, according to Ref. 31Wishart D.S. Bigam C.G. Yao J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2053) Google Scholar.Analysis of Thiol Groups and Redox Titration of CP12-2—Protein thiol analyses and redox titrations were performed with pure CP12-2 in 100 mm Tricine-NaOH, pH 7.9. Redox titration experiments were performed with 70 μm CP12-2 incubated for 3 h at 25 °C with variable ratios of reduced and oxidized DTT (20 mm total concentration) in a final volume of 500 μl. After incubation, samples were desalted with PD10 columns (GE Healthcare) equilibrated with 100 mm Tricine-NaOH, pH 7.9. In order to avoid any possible DTT contamination, only the first 2 ml of eluted samples were collected. Control experiments were performed under the same conditions but in the absence of CP12-2. Absorbance at 280 and 412 nm was recorded immediately before and after the addition of 0.5 mm 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). The number of solvent-accessible thiol groups under different redox conditions was calculated from the ratio between the absorbance at 412 nm (molar extinction coefficient of 14,150 m–1 for 2-nitro-5-thiobenzoate (thiolate) dianion) (32Conway M.E. Poole L.B. Hutson S.M. Biochemistry. 2004; 43: 7356-7364Crossref PubMed Scopus (62) Google Scholar) and the absorbance at 280 nm (molar extinction coefficient of 8,370 m–1 for CP12-2) (7Marri L. Trost P. Pupillo P. Sparla F. Plant Physiol. 2005; 139: 1433-1443Crossref PubMed Scopus (65) Google Scholar).Redox titration results were fit by nonlinear regression (CoHort Software) to the Nernst equation for two redox components (26Sparla F. Pupillo P. Trost P. J. Biol. Chem. 2002; 277: 44946-44952Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Midpoint redox potentials are reported as average values ± S.D. of triplicate experiments.Isothermal Titration Calorimetry (ITC)—Calorimetric measurements were carried out using a VP-ITC MicroCalorimeter (MicroCal Inc., Northampton, MA). Each experiment was performed at a constant temperature of 30 °C and consisted of 25 injections of 10-μl aliquots, repeated every 200 s. All samples were degassed by stirring under vacuum before use. Heat of dilution, measured by control experiments in which samples were injected into a buffer-filled cell, was subtracted. Signals recorded in each experiment were integrated using OriginPro 7.5 software supplied with the instrument. The thermodynamic binding parameters (dissociation constant (KD), variations of enthalpy (ΔH), Gibbs free energy (ΔG), entropy (ΔS), and the number of binding sites (n)) were obtained by nonlinear regression of the integrated heat plots, according to the “one set of sites” model of the software.Calorimetric titrations of binary complex formation were carried out with 15 μm oxidized CP12-2 in 25 mm potassium phosphate, 0.2 mm NAD, pH 7.5, in both sample and reference cells, whereas the syringe was filled with 52 μm A4-GAPDH in the same buffer. Although CP12-2 behaved as a ligand, it was not filled in the syringe because of its very high heat of dilution. The presence of ligand (CP12-2) in the cell and macromolecule (A4-GAPDH) in the syringe was taken into account in the elaboration of primary data.Calorimetric titrations of ternary complex formation were performed with 5 μm preformed binary complex in 25 mm potassium phosphate, 0.2 mm NAD, pH 7.5, in sample and reference cells, whereas the syringe was filled with 70 μm PRK dissolved in the same buffer. Thermodynamic parameters of binary and ternary complex formation are reported as average values ± S.D. of triplicate experiments.Dynamic Light Scattering (MALS-QELS)—Purified single proteins and preformed binary and ternary complexes were analyzed by size exclusion chromatography connected to a multiangle light scattering (MALS) equipped with QELS module (quasielastic light scattering) for RH measurements. 100-μl samples were loaded on a Superdex 200HR column (GE Health-care) equilibrated in 25 mm potassium phosphate, pH 7.5, 1 mm EDTA, 150 mm KCl, with 0.2 mm NAD (for A4-GAPDH, binary, and ternary complexes) or without NAD (for PRK and CP12-2). A constant flow rate of 0.6 ml min–1 was applied. Elution profiles were detected by an Optilab rEX interferometric refractometer and a Dawn EOS multiangle laser light-scattering system at 690 nm (Wyatt Technology Corp.). Data acquisition and processing were carried out using ASTRA 5.1.9.1 software (Wyatt Technology). Determination of molecular masses and hydrodynamic radii are reported as mean values ± S.D. of duplicate experiments.RESULTSCP12-2 of A. thaliana Is Intrinsically Unstructured and Monomeric—NMR analysis of CP12-2 in the oxidized state revealed that most of the amide proton resonances are localized between 8.5 and 8.0 ppm, the so-called random coil region, strongly suggesting that CP12-2 is mainly unstructured (Fig. 1). Only a few residues located at the C terminus exhibit amide chemical shifts outside the random coil range, indicating structuration (33Yao J. Dyson H.J. Wright P.E. FEBS Lett. 1997; 419: 285-289Crossref PubMed Scopus (147) Google Scholar). Consistent with the importance of disulfide bridges in this respect, the reduction of oxidized CP12-2 by DTT led to typical random coil signals (not shown), as previously described for Chlamydomonas CP12 (20Graciet E. Gans P. Wedel N. Lebreton S. Camadro J.M. Gontero B. Biochemistry. 2003; 42: 8163-8170Crossref PubMed Scopus (98) Google Scholar).The calculated molecular mass of recombinant CP12-2 (after proteolytic cleavage of the His tag) was 8.7 kDa. Although oxidized CP12-2 behaves as a protein of 29 kDa in size exclusion chromatography (7Marri L. Trost P. Pupillo P. Sparla F. Plant Physiol. 2005; 139: 1433-1443Crossref PubMed Scopus (65) Google Scholar), MALS-QELS analysis of the protein eluted from the size exclusion column yielded a molecular mass of 9 ± 1 kDa, conclusively demonstrating that under native conditions, CP12-2 is a monomer (Table 1).TABLE 1Determination of molecular mass (m) and hydrodynamic radii (RH) of purified single proteins and reconstituted complexes by MALS-QELSSampleCalculatedMeasuredmRHaThe theoretical hydrodynamic radius RH for a spherical protein of a given Mr was calculated on the basis of the empirical equation Mr = 4/3NA(RHf/f0)3/Vk, where NA is Avogadro's number, f/f0 is the ratio of frictional coefficients (set to 1.2 for spherical proteins), and Vk is the partial volume set to 0.73 for a spherical protein (45).mRHkDanmkDanmCP12-28.71.639 ± 1NAbThe experimental RH of CP12-2 was close to the lower detection limit of the QELS module.A4-GAPDH148.8cSince chromatographic runs were performed in the presence of 0.2 mm NAD, molecular masses were calculated on the assumption that each A4-GAPDH tetramer bound four NAD molecules.4.21146 ± 23.9 ± 0.3PRK77.63.3985 ± 73.3 ± 0.3(A4-GAPDH) + (CP12-2)166.2cSince chromatographic runs were performed in the presence of 0.2 mm NAD, molecular masses were calculated on the assumption that each A4-GAPDH tetramer bound four NAD molecules.,dCalculated molecular mass for a binary complex with stoichiometry (A4-GAPDH)-(CP12-2)2.4.36170 ± 144.3 ± 0.5(A4-GAPDH) + (CP12-2) + (PRK)487.7cSince chromatographic runs were performed in the presence of 0.2 mm NAD, molecular masses were calculated on the assumption that each A4-GAPDH tetramer bound four NAD molecules.,eCalculated molecular mass for a ternary complex with stoichiometry ((A4-GAPDH)-(CP12-2)2-(PRK))2.6.25498 ± 67.0 ± 0.1a The theoretical hydrodynamic radius RH for a spherical protein of a given Mr was calculated on the basis of the empirical equation Mr = 4/3NA(RHf/f0)3/Vk, where NA is Avogadro's number, f/f0 is the ratio of frictional coefficients (set to 1.2 for spherical proteins), and Vk is the partial volume set to 0.73 for a spherical protein (45Michels J. Geyer A. Mocanu V. Welte W. Burlingame A.L. Przybylski M. Protein Sci. 2002; 11: 1565-1574Crossref PubMed Scopus (10) Google Scholar).b The experimental RH of CP12-2 was close to the lower detection limit of the QELS module.c Since chromatographic runs were performed in the presence of 0.2 mm NAD, molecular masses were calculated on the assumption that each A4-GAPDH tetramer bound four NAD molecules.d Calculated molecular mass for a binary complex with stoichiometry (A4-GAPDH)-(CP12-2)2.e Calculated molecular mass for a ternary complex with stoichiometry ((A4-GAPDH)-(CP12-2)2-(PRK))2. Open table in a new tab CP12-2 Redox Properties—To gain insight into the redox properties of CP12-2 cysteines, redox titrations were performed in the presence of DTNB as a probe to reveal free protein thiols under varying redox conditions. Fully reduced/oxidized samples were obtained following equilibration with 20 mm reduced or oxidized DTT, which was then removed by desalting. Although reduced CP12-2, whose amino acid sequence includes four Cys residues, was found by DTNB titration to contain four reactive thiols (4.5 ± 0.5), oxidized CP12-2 had none (–0.3 ± 0.1), indicating that both CP12-2 disulfides could be redox-titrated by DTT plus DTNB. Data from redox titrations of purified CP12-2 were therefore fitted to a Nernst equation for two different thiol/disulfide equilibria, equally contributing to the total redox response. At pH 7.9, the midpoint redox potentials (Em,7.9) of CP12-2 disulfides were estimated as –326 ± 2 and –352 ± 6 mV, respectively (Fig. 2).FIGURE 2Redox ti" @default.
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• W2054523600 title "Spontaneous Assembly of Photosynthetic Supramolecular Complexes as Mediated by the Intrinsically Unstructured Protein CP12" @default.
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