title: NanoESI mass spectrometry of Rubisco and Rubisco activase structures and their interactions with nucleotides and sugar phosphates.
abstract: Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is the protein that is responsible for the fixation of carbon dioxide in photosynthesis. Inhibitory sugar phosphate molecules, which can include its substrate ribulose-1,5-bisphosphate (RuBP), can bind to Rubisco catalytic sites and inhibit catalysis. These are removed by interaction with Rubisco activase (RA) via an ATP hydrolytic reaction. Here we show the first nanoESI mass spectra of the hexadecameric Rubisco and of RA from a higher plant (tobacco). The spectra of recombinant, purified RA revealed polydispersity in its oligomeric forms (up to hexamer) and that ADP was bound. ADP was removed by dialysis against a high ionic strength solution and nucleotide binding experiments showed that ADP bound more tightly to RA than AMP-PNP (a non-hydrolysable ATP analog). There was evidence that there may be two nucleotide binding sites per RA monomer. The oligomerization capacity of mutant and wild-type tobacco RA up to hexamers is analogous to the subunit stoichiometry for other AAA+ enzymes. This suggests assembly of RA into hexamers is likely the most active conformation for removing inhibitory sugar phosphate molecules from Rubisco to enable its catalytic competency. Stoichiometric binding of RuBP or carboxyarabinitol bisphosphate (CABP) to each of the eight catalytic sites of Rubisco was observed.
authors.affiliation: School of Chemistry, University of Wollongong, New South Wales, 2522, Australia.
authors.forename: Michelle J
authors.initials: MJ
authors.lastname: Blayney
authors.forename: Spencer M
authors.initials: SM
authors.lastname: Whitney
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Beck
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D018832:Molecular Chaperones; D008970:Molecular Weight; D009154:Mutation; D036103:Nanotechnology; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014026:Nicotiana
chemical_list: D018832:Molecular Chaperones; D009711:Nucleotides; D010940:Plant Proteins; C054765:rca protein, plant; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D012273:Ribulose-Bisphosphate Carboxylase