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 A(4)), 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 A(4)-GAPDH (K(D) = 0.18 microm) to form a binary complex of 170 kDa with (A(4)-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 (K(D) = 0.17 microm) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A(4)-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 A(4)-GAPDH and PRK during darkness in photosynthetic tissues.
Highlights
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
Oxicarried out with 15 M oxidized CP12-2 in 25 mM potassium dized CP12-2 behaves as a protein of 29 kDa in size exclusion phosphate, 0.2 mM NAD, pH 7.5, in both sample and reference chromatography [7], MALS-QELS analysis of the protein cells, whereas the syringe was filled with 52 M A4-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in eluted from the size exclusion column yielded a molecular mass the same buffer
CP12 is a widespread regulatory protein of oxygenic photosynthetic organisms that contributes to the regulation of carbon metabolism by producing supramolecular complexes with two enzymes, GAPDH and PRK, accounting for most of the energetic needs of the Calvin cycle [2, 3]
Summary
Protein 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 [7]. After 16 –18 h of incubation at 4 °C, the sample was desalted in 25 mM potassium phosphate buffer, pH 7.0, and concentrated. Redox titration experiments were performed with 70 M 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 l. Heat of dilution, measured by control experiments in which samples were injected into a buffer-filled cell, was subtracted. Calorimetric titrations of ternary complex formation were performed with 5 M 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 M PRK dissolved in the same buffer. Thermodynamic parameters of binary and ternary complex formation are reported as average values Ϯ S.D. of triplicate experiments. Data shown are mean values Ϯ S.D. of duplicate runs
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