Exploration of CP12 conformational changes and of quaternary structural properties using electrospray ionization traveling wave ion mobility mass spectrometry

Kaaki, Wassim; Woudstra, Mireille; Gontero, Brigitte; Halgand, Frédéric

doi:10.1002/rcm.6442

Cited by 14 publications

(20 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Based on the similar fluorescence brightness found for CP12 mutant proteins alone and for the sub‐complexes, only one monomer of CP12C31S or CP12C75S bound to GAPDH and PRK respectively. The mutant CP12C31S behaves as CP12 locked in a reduced state and therefore this result is in agreement with previous results where only one molecule of reduced CP12 was bound to GAPDH while two molecules of oxidized CP12 were bound to tetrameric GAPDH . The present study also indicates that the ratio 1 : 4 of CP12 to PRK is sufficient to form the CP12C75S–PRK complex; with a K D of 1.6 μ m determined by surface plasmon resonance (SPR) and the experimental conditions used in this report, one would expect about 20% of complex formation between CP12 and PRK.…”

Section: Discussionsupporting

confidence: 93%

“…C). For all experiments, His‐tagged CP12 proteins were used as it was shown that wild‐type His‐tagged CP12 behaves like the native protein, indicating that the His tag does not interfere in our experiments . Numbering of residues, however, was based on the protein without the tag, with SGQPA being the first residues (http://genome.jgi.doe.gov/cgi-bin/dispGeneModel?db=Chlre4&id=148487).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Conformational modulation and hydrodynamic radii of CP12 protein and its complexes probed by fluorescence correlation spectroscopy

et al. 2014

Self Cite

View full text Add to dashboard Cite

Light/dark regulation of the Calvin cycle in oxygenic photosynthetic organisms involves the formation and dissociation of supramolecular complexes between CP12, a nuclear-encoded chloroplast protein, and the two enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.13) and phosphoribulokinase (PRK) (EC 2.7.1.19). Despite the high importance of understanding the structural basis of the interaction of CP12 with GAPDH and PRK to investigate the regulation of the Calvin cycle, information is still lacking about the structural remodulation of CP12 and its complex formation. Here, we characterize the diffusion dynamics and hydrodynamic radii of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH and PRK using fluorescence correlation spectroscopy experiments. We quantify a hydrodynamic radius of 3.4 AE 0.2 nm for the CP12 protein with an increase up to 5.2 AE 0.3 nm upon complex formation with GAPDH and PRK. In addition, unfolding experiments reveal a 1.6-and 2.0-fold increase respectively of the hydrodynamic radii for the N-terminal and C-terminal cysteine CP12 mutant proteins compared with their native folded structures. The different behavior of the CP12 mutant proteins during hydrophobic collapse transition is a direct clue to different structural orientations of the CP12 mutant proteins. These different structures are expected to facilitate the binding of either GAPDH or PRK during binary complex and ternary complex formation. Structured digital abstractGAPDH, CP12 and PRK physically interact by fluorescence correlation spectroscopy (View interaction) CP12 and PRK bind by fluorescence correlation spectroscopy (View interaction) GAPDH and CP12 bind by fluorescence correlation spectroscopy(View interaction)

show abstract

Section: Discussionsupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Conformational modulation and hydrodynamic radii of CP12 protein and its complexes probed by fluorescence correlation spectroscopy

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…To reconstitute this complex, a stoichiometry of 2 tetrameric GAPDH, 2 dimeric PRK and 4 monomeric CP12 (molar concentration) were used as described by Avilan et al and Kaaki et al 27,39 Native PAGE followed by immunoblotting experiments showed that the ternary complex was formed with the wild type protein as well as with the C16S, C55S and C61S mutants (Fig. To reconstitute this complex, a stoichiometry of 2 tetrameric GAPDH, 2 dimeric PRK and 4 monomeric CP12 (molar concentration) were used as described by Avilan et al and Kaaki et al 27,39 Native PAGE followed by immunoblotting experiments showed that the ternary complex was formed with the wild type protein as well as with the C16S, C55S and C61S mutants (Fig.…”

Section: Involvement Of Cys243 and Cys249 In The Gapdh-cp12-prk Complmentioning

confidence: 99%

Phosphoribulokinase from Chlamydomonas reinhardtii: a Benson–Calvin cycle enzyme enslaved to its cysteine residues

et al. 2015

Self Cite

View full text Add to dashboard Cite

Phosphoribulokinase (PRK) in the green alga Chlamydomonas reinhardtii is a finely regulated and well-studied enzyme of the Benson-Calvin cycle. PRK can form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small chloroplast protein CP12. This study aimed to determine the molecular determinants on PRK involved in the complex and the mechanism of action of a recently described novel regulation of PRK that involves glutathionylation. A combination of mass spectrometry, mutagenesis and activity analyses showed that Cys16, besides its role as the binding site of ATP, was also the site for S-glutathionylation. Previous kinetic analysis of the C55S mutant showed that in the oxidized inactive form of PRK, this residue formed a disulfide bridge with the Cys16 residue. This is the only bridge reported for PRK in the literature. Our data show for the first time that a disulfide bridge between Cys243 and Cys249 on PRK is required to form the PRK-GAPDH-CP12 complex. These results uncover a new mechanism for the PRK-GAPDH-CP12 formation involving a thiol disulfide exchange reaction with CP12 and identify Cys16 of PRK as a target of glutathionylation acting against oxidative stress. Although Cys16 is the key residue involved in binding ATP and acting as a defense against oxidative damage, the formation of the algal ternary complex requires the formation of another disulfide bridge on PRK involving Cys243 and Cys249.

show abstract

“…Although it is unusual for an intrinsically disordered protein to migrate according to its actual molecular mass on a size‐exclusion gel, a similar result has been found for a well‐characterized CP12 from C. reinhardtii that elutes at c . 30 kDa when alone (Kaaki et al ., ) but at 8.5 kDa immediately after dissociation from the GAPDH–CP12 complex (B. Gontero & C. Puppo, unpublished). Incubation of GAPDH alone with NADPH, DTT or NADP had no effect on its activity and all progress curves were similar (Fig.…”

Section: Resultsmentioning

confidence: 97%

Glyceraldehyde‐3‐phosphate dehydrogenase is regulated by ferredoxin‐NADP reductase in the diatom Asterionella formosa

et al. 2014

Self Cite

View full text Add to dashboard Cite

SummaryDiatoms are a widespread and ecologically important group of heterokont algae that contribute c. 20% to global productivity. Previous work has shown that regulation of their key Calvin cycle enzymes differs from that of the Plantae, and that in crude extracts, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be inhibited by nicotinamide adenine dinucleotide phosphate reduced (NADPH) under oxidizing conditions.The freshwater diatom, Asterionella formosa, was studied using enzyme kinetics, chromatography, surface plasmon resonance, mass spectrometry and sequence analysis to determine the mechanism behind this GAPDH inhibition.GAPDH interacted with ferredoxin-nicotinamide adenine dinucleotide phosphate (NADP) reductase (FNR) from the primary phase of photosynthesis, and the small chloroplast protein, CP12. Sequences of copurified GAPDH and FNR were highly homologous with published sequences. However, the widespread ternary complex among GAPDH, phosphoribulokinase and CP12 was absent. Activity measurements under oxidizing conditions showed that NADPH can inhibit GAPDH-CP12 in the presence of FNR, explaining the earlier observed inhibition within crude extracts.Diatom plastids have a distinctive metabolism, including the lack of the oxidative pentose phosphate pathway, and so cannot produce NADPH in the dark. The observed down-regulation of GAPDH in the dark may allow NADPH to be rerouted towards other reductive processes contributing to their ecological success.

show abstract

Exploration of CP12 conformational changes and of quaternary structural properties using electrospray ionization traveling wave ion mobility mass spectrometry

Cited by 14 publications

References 45 publications

Conformational modulation and hydrodynamic radii of CP12 protein and its complexes probed by fluorescence correlation spectroscopy

Conformational modulation and hydrodynamic radii of CP12 protein and its complexes probed by fluorescence correlation spectroscopy

Phosphoribulokinase from Chlamydomonas reinhardtii: a Benson–Calvin cycle enzyme enslaved to its cysteine residues

Glyceraldehyde‐3‐phosphate dehydrogenase is regulated by ferredoxin‐NADP reductase in the diatom Asterionella formosa

Contact Info

Product

Resources

About