Self Assembly of Rubisco Activase Monitored by Fluorescence Fluctuation Spectroscopy

RuBisCO Activase (Rca) is a protein that belongs to the AAA+ family of the ATPases which utilizes the energy derived from ATP hydrolysis to release firmly bound sugar phosphates from the active site of the Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The challenge associated with structure and functional investigation of Rca can be attributed to its exceptionally low thermo-stability, high degree of size polydispersity and tendency toward subunit aggregation.In this work we have successfully employed fluorescence fluctuation methods to study the nucleotide-dependent stoichiometry of fluorescently tagged Rca for a wide range of concentrations. Our results show a stepwise assembly pathway of Rca under different assay conditions. In presence of Mg+2 -ADP, the oligomerization state of Rca is largely dominated by monomers at concentrations below 0.5 μM. The state of oligomerization gradually changes in steps of two subunits. The most probable model for this assembly supports the dissociation coefficients of ∼4, 1, 1 μM for the monomer-dimer, dimer-tetramer and tetramer-hexamer equlibria respectively. Continued assembly at even higher concentrations suggests self association through the formation of spiral arrangements that grow along the helical axis.