title: Biophysical characterization of higher plant Rubisco activase.
abstract: Rubisco activase (Rca) is a chaperone-like protein of the AAA+ family, which uses mechano-chemical energy derived from ATP hydrolysis to release tightly bound inhibitors from the active site of the primary carbon fixing enzyme ribulose 1,5-bisphosphate oxygenase/carboxylase (Rubisco). Mechanistic and structural investigations of Rca have been hampered by its exceptional thermolability, high degree of size polydispersity and propensity towards subunit aggregation. In this work, we have characterized the thermal stability and self-association behavior of recombinant Rca preparations, and have developed ligand screening methods. Thermal denaturation profiles generated by circular dichroism indicate that creosote and tobacco short-form Rcas are the most stable proteins examined, with an estimated mid-point temperature of 45-47°C for protein denaturation. We demonstrate that ADP provides a higher degree of stabilization than ATP, that magnesium ions have a small stabilizing effect on ATP-bound, but a significant destabilizing effect on ADP-bound Rca, and that phosphate provides weak stabilization of the ADP-bound form of the protein. A dimeric species was identified by size-exclusion chromatography, suggesting that the two-subunit module may comprise the basic building block for larger assemblies. Evidence is provided that chromatographic procedures reflect non-equilibrium multimeric states. Dynamic light scattering experiments performed on nucleotide-bearing Rca support the notion that several larger, highly polydisperse assembly states coexist over a broad concentration range. No significant changes in aggregation are observed upon replacement of ADP with ATP. However, in the absence of nucleotides, the major protein population appears to consist of a monodisperse oligomer smaller than a hexamer.
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA.
authors.forename: J Nathan
authors.initials: JN
authors.lastname: Henderson
authors.forename: Suratna
authors.initials: S
authors.lastname: Hazra
authors.forename: Alison M
authors.initials: AM
authors.lastname: Dunkle
authors.forename: Michael E
authors.initials: ME
authors.lastname: Salvucci
authors.forename: Rebekka M
authors.initials: RM
authors.lastname: Wachter
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D002942:Circular Dichroism; D032442:Larrea / Q000737:chemistry*; D018832:Molecular Chaperones / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D014026:Nicotiana / Q000737:chemistry*
chemical_list: D018832:Molecular Chaperones; D010940:Plant Proteins; C054765:rca protein, plant; D000255:Adenosine Triphosphate